Experimental Streptococcus iniae infection of hybrid striped bass (Morone chrysops×Morone saxatilis) and tilapia (Oreochromis niloticus) by nares inoculation

Aquaculture 189 Ž2000. 197–210 www.elsevier.nlrlocateraqua-online

Experimental Streptococcus iniae infection of hybrid striped bass žMorone chrysops= Morone saxatilis / and tilapia žOreochromis niloticus / by nares inoculation Joyce J. Evans a,) , Craig A. Shoemaker b, Phillip H. Klesius b a

United States Department of Agriculture, Agricultural Research SerÕice, Aquatic Animal Health Research Laboratory, 300 Washington AÕe., Chestertown, MD 21620, USA b P.O. Box 952, Auburn, AL 36830-0952, USA Received 18 October 1999; received in revised form 27 March 2000; accepted 27 March 2000

Abstract Streptococcus iniae infection was evaluated in hybrid striped bass and tilapia following experimental, bilateral inoculation of a virulent isolate of S. iniae onto their eyes or into their nares. Inoculation of eyes did not result in mortality or disease signs in either species. In contrast, mortality and disease signs were observed in both species following nares inoculation. Hybrid striped bass were susceptible to 4.8 = 10 3 S. iniae colony forming units ŽCFU., an inoculum 100 times lower than that affecting tilapia. We observed decreased mortality of hybrid striped bass administrated 4.8 = 10 5 CFU dose of S. iniae. Following S. iniae infection, the disease signs observed in hybrid striped bass and tilapia were erratic, serpentine, rising and falling, and head-up swimming, slow acceptance or refusal of food, lethargy and darkened skin. We did not observe exophthalmia, eye opacity or body curvature. We were able to recover S. iniae from either nares, eyes, brains, or head kidney of 87.5% and 66.7% of the morbid hybrid striped bass and tilapia, respectively. Both hybrid striped bass and tilapia produced significant anti-streptococcal titers 14 days post nares inoculation with 4.8 = 10 3 CFU of S. iniae. Successful experimental S. inaie infection of fish following nares inoculation suggest that nares may be a potential route of S. iniae infection in cultured fish species. This route of experimental infection for S. iniae has not been previously described in fish. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Infectivity; Streptococcus iniae; Hybrid striped bass; Tilapia; Nare; Eye; Mortality; Disease signs

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Corresponding author. Tel.: q1-410-778-2120; fax: q1-410-778-4399. E-mail address: [email protected] ŽJ.J. Evans..

0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 0 . 0 0 3 7 6 - 8

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1. Introduction Streptococcal disease is the cause of significant economic losses to the world aquaculture industry ŽBoomker et al., 1979; Perera et al., 1994, 1997; Stoffregen et al., 1996; Shoemaker and Klesius, 1997.. These losses have been estimated to exceed US $150 million annually ŽShoemaker and Klesius, 1997.. Streptococcal disease has been reported in numerous cultured fish ŽKitao, 1993.. Although the principal economic impact from this bacterial disease is associated with aquaculture, death in many species of feral fish have also been reported ŽPlumb et al., 1974; Cook and Lofton, 1975; Baya et al., 1990. but the streptococcal species was not identified. Disease losses in feral fish may have a significant economic impact to the commercial and sport fishery industry although difficult to quantitate. Streptococcus sp. have been isolated from seawater and bottom mud of pen-culture areas and contaminated fish food ŽKusuda and Kawai, 1982; Minami, 1979.. Streptococcus sp. have been shown to gain entry into fish by ingestion, injured skin, and experimental injection. Robinson and Meyer Ž1966. demonstrated that ingestion of moribund fish caused streptococcal disease in golden shiners Ž Notemigonous crysoleucas .. Ingestion of streptococcal contaminated fish food caused streptococcal disease in yellowtail ŽMinami, 1979.. Rasheed and Plumb Ž1984. demonstrated gulf killifish Ž Fundulus grandis . are susceptible through injury of the skin from water contaminated with Streptococcus sp. Entry by intraperitoneal and intramuscular injection has also caused streptococcal disease in fish ŽHoshina et al., 1958; Robinson and Meyer, 1966; Cook and Lofton, 1975; Ohnishi and Jo, 1981; Iida et al., 1986.. Although it is clear that these routes of infection result in streptococcal disease, experimental routes of infection from more recently characterized streptococcal species such as Streptococcus iniae have been poorly studied. S. iniae has been identified as one of the principal etiologic agents of streptococcal disease in cultured hybrid striped bass Ž Morone chrysops= Morone saxatilis . ŽStoffregen et al., 1996; Perera et al., 1997., rabbitfish Ž Siganus canaliculatus. ŽYuasa et al., 1999., tilapia Ž Oreochromis sp.. ŽPerera et al., 1994; Eldar et al., 1995; Bowser et al., 1998; Perera et al., 1997; Klesius et al., 1999., rainbow trouts Ž Oncorhynchus mykiss, Oncorhynchus kisutch. ŽEldar et al., 1995. red drum Ž Sciaenops ocellatus . ŽEldar et al., 1999. and barramundi Ž Lates calcarifer . ŽBromage et al., 1999.. Zlotkin et al. Ž1998. reported S. iniae infections in populations of cultured gilthead sea bream Ž Sparus aurata., European sea bass Ž Dicentrarchus labrax ., and wild spine foot Ž Siganus riÕulatus. living in the same proximity and suggested the possible transmission of S. iniae from wild fish to cultured fish. This organism is a Gram-positive, aerobic, coccus-shaped bacterium that has been isolated from brain, eye, or kidneys ŽStoffregen et al., 1996; Bowser et al., 1998; Perera et al., 1998. of naturally infected fish. A potential source of S. iniae infection is water contaminated with S. iniae infected morbid and dead fish. Entry of S. iniae through the nares andror the eyes of cultured fish may be a likely route of infection from contaminated water, although the nares are not a commonly cultured site. Investigations of routes of infection, for at least one other bacterial fish pathogen, Edwardsiella ictaluri, by contact with infected fish ŽShotts et al., 1986., water immersion ŽNewton et al., 1989., and nares inoculation ŽMorrison and

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Plumb, 1994. were shown to cause disease signs, mortality, and brain pathology in channel catfish Ž Ictalurus punctatus .. In this study, we present the results of mortality and recovery of S. iniae from fish, disease signs, and anti-streptococcal antibody responses following experimental inoculation of the nares and eyes of hybrid striped bass and tilapia with a single virulent isolate of S. iniae.

2. Materials and methods 2.1. Fish Hybrid striped bass Žmean weight and length 13.1 g and 10.3 cm. and tilapia Žmean weight and length 22.7 g and 11.1 cm. were acclimated in flow-through 57 l glass aquaria supplied with 0.5 lrh de-chlorinated water for 10 days prior to experiments. The hybrid striped bass were obtained from Keo Farms ŽKeo, AR. and the tilapia were from stocks maintained at the ARS, USDA, Aquatic Animal Health Research Laboratory ŽAuburn, AL.. A light and dark period of 12:12 h was maintained and aeration was supplied through an air stone. The fish were fed daily to satiation with Aquamax Grower 400 ŽBrentwood, MO.. To verify the S. iniae-free status of the fish, samples were obtained for bacterial culture by passing an inoculation loop into nare, eye, brain, and kidney. Samples were streaked directly on sheep blood agar that were incubated at 278C for 24–48 h. S. iniae was not isolated from randomly selected hybrid striped bass Ž4. and tilapia Ž5.. 2.2. Water quality eÕaluation The dissolved oxygen, temperature, pH, salinity, hardness, ammonia, and nitrites were measured daily. The dissolved oxygen, temperature, and salinity were measured using a YSI 85 oxygen conductivity, salinity, and temperature meter ŽYellow Spring Instrument, Yellow Springs, OH.. The pH, hardness, ammonia and nitrites were determined using the Fresh Water Aquaculture Kit Model AG-2 ŽLaMotte., Chestertown, MD.. In experiments I and II, the mean " standard deviation of dissolved oxygen Žmgrl. was 5.4 " 0.449 and 5.8 " 0.512, temperature Ž8C. was 26.2 " 0.799 and 26.4 " 0.449, pH was 7.2 " 0.0 and 7.1 " 0.253, salinity Ž%. was 0.1 " 0.00 and 0.1 " 0.00, and hardness was 110 " 10.0 and 110 " 10.0, respectively. Ammonia and nitrite concentrations Žmgrl. were 0 in both experiments. 2.3. Bacteria S. iniae strain ARS-98-60, originally isolated from a hybrid striped bass with natural streptococcal disease was used to infect fish. The isolate was identified as S. iniae by its white petite colony and coccus cell morphology, Gram-positive stain, negative catalase production, b hemolytic activity in sheep’s blood agar, lack of motility, inability to produce acetyl methyl carbinol ŽVoges–Proskauer test., variable growth in 6.5% NaCl, ability to hydrolyze starch, pyrrolydonlarylamidase ŽPYR. and leucine aminopeptidase

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ŽLAP. and inability to hydrolyze sodium hippurate, tolerance to bile-esculin, inability to ferment sorbitol and to produce gas in Lactobacillus, Mann, Rogosa and Sharpe ŽMRS. broth, variable ability to deaminate arginine, ability to enhance b-lysin produced by Staphylococcus aureus SS-695 CDC strain ŽCAMP test. and susceptibility to vancomycin and resistance to bacitracin ŽShoemaker and Klesius, 1997.. The isolate was grown in tryptic soy broth ŽTSB, Difco Laboratories, Sparks, MD. for 24 h at 278C and then the culture isolate was frozen in 0.2 ml aliquots at y708C. The infectious isolate used in this study was prepared by inoculating 250 ml of TSB in 500 ml culture flask with a thawed aliquot of the frozen isolate. Following, stationary incubation for 24 h at 278C, the culture was adjusted spectrophotometrically to an optical density of 1.2 absorbancy units at 540 nm to give a S. iniae concentration of 2.4 = 10 8 colony forming units ŽCFU.rml. The doses of S. iniae used in this study were then prepared by dilution in TSB or concentrated by centrifugation, 10–15 min prior to use and the CFU were later confirmed by plate counts. Colony counts were performed by standard methods and visually using a colony counter and a light box ŽBel-Art products, Pequannock, NJ.. Sterile TSB was used as the control. 2.4. Nares and eyes experimental inoculation Hybrid striped bass and tilapia were anesthetized in 3 l of water containing 25 mg tricaine methanesulfonaterl. Fish were removed, placed on moist towels, and then inoculated using micropipet Žtip diameter, - 50 mm. carefully inserted into the anterior incurrent nare. The nare was slowly instilled with 10 ml aliquot of the culture suspension or control TSB. Special care was taken not to injure the olfactory mucosa during the inoculation procedure. To inoculate the eye, the tip of the micropipet was carefully held slightly above the surface of the eye and then 10 ml of the culture suspension or control TSB was slowly dropped onto the surface of the eye. These procedures were repeated to inoculate both nares or both eyes of each fish. The inoculated fish were carefully positioned upright on moist paper towels and allowed to recover for 5 min. The fish were then placed back in their respective tanks for complete recovery in 5–10 min. 2.5. Experimental design The study was conducted in two sequential experiments using both hybrid striped bass and tilapia. In experiment I, 120 hybrid striped bass and 120 tilapia were assigned to one of the eight groups of 15 fish each. Two groups for each species were designated as control and the remaining six groups for each species were inoculated with 4.8 = 10 3, =10 4 , or =10 5 CFU into either their nares or onto their eyes. The control groups were administered TSB only into their nares or eyes. Groups were not replicated in this experiment. In experiment II, 240 hybrid striped bass and 240 tilapia were assigned to one of four groups in triplicate with 20 fish per replicate. One group of hybrid striped bass was designated as control and the nares of fish in the remaining groups were inoculated with 2.6 = 10 4 , 5.2 = 10 4 , or 1.0 = 10 5 CFU. For tilapia, one group was designated as control and the nares of the remaining groups were inoculated with

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1.4 = 10 7, =10 8 , or =10 9 CFU. The nares of hybrid striped bass and tilapia in the control groups received TSB only. After inoculation, fish were monitored immediately and twice daily for disease signs and mortality and water quality was monitored for a 14-day study period. 2.6. Bacteriologic sample collection and eÕaluation Dead and moribund fish were removed twice a day for the duration of the 14-day study period. At postmortem examination, bacterial samples were obtained aseptically from nare, eye Žretro-orbitally., brain, and anterior kidney sites. Specimens were cultured directly onto sheep blood agar media purchased from Remel ŽLenexa, KS. at 278C for 24–48 h. Beta-hemolytic, catalase-negative, and Gram-stained positive coccus colonies were subcultured onto sheep blood agar and then bacteriologically and biochemically identified as S. iniae according to tests described by Shoemaker and Klesius Ž1997.. At the end of the 14-day study period, surviving fish in experiment I were also bacteriologically assayed and isolates were identified as S. iniae. 2.7. Blood collection and serologic eÕaluation In experiment I, blood samples were collected from 4–7 surviving hybrid striped bass and tilapia from each treatment group. Blood was drawn from caudal vein into micro-capillary tube, allowed to clot for 1 h at 258C, and then centrifuged at 1000 = g for 10 min. Serum was collected and stored in a plastic-capped tube at y708C until assayed for antibody titer. Streptococcal antibody titer in serum was determined by use of a microtitration agglutination test. Briefly, each well of a 96-round well microtitration plate was inoculated with 50 ml of phosphate-buffered saline ŽPBS. solution ŽpH 7.2–7.4. and then 50 ml of hybrid striped bass or tilapia serum was added to the first well of each row, mixed and then 50 ml of diluted serum was serially diluted in twofold steps into the remaining wells. Doubling dilutions of positive and negative sera were included on every plate as controls. To each well, 50 ml of S. iniae ARS-98-60 cell suspension was added and mixed. The plate was covered and incubated in humidified air at 258C for 18 h. The highest serum dilution that showed a circular diffuse button with fuzzy edges at the bottom of the well was considered a positive reaction and a circular compact cell button was considered to be a negative reaction. Antibody titer was expressed as the log base10 . 2.8. Mortality and disease signs (behaÕioral and morphological) eÕaluation Mortality and disease signs were recorded on a daily checklist. Behavioral characteristics of S. iniae disease were: Ž1. location of fish in the aquarium Žfish surface swimming or stationary at the bottom of the water column.; Ž2. swimming pattern Žfish stationary, fish either swimming on their side, serpentine swimming, head up swimming or frequently sinking and rising.; Ž3. feeding response Žfish slowly accepting or refusing food.; Ž4. activityrexcitability Žfish either hyperactive, lethargic, or unresponsive; and

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Ž5. respiration Žfish had opercular rapidity or gasping.. Morphology characteristics of S. iniae disease included fish with either dark skin coloration, eye opacity, or body curvature. 2.9. Statistical analysis Mortality data from experiment I were not analyzed statistically due to the absence of treatment replication. The significance between log base10 antibody titers among treatment groups in experiment I and the significant differences between mean mortality among the treatment groups in experiment II were determined by one way analysis of variance and Duncan’s multiple-range test ŽSAS Institute, 1997.. Differences in antibody titer and mean mortality between treatment groups were significant at P - 0.05.

3. Results 3.1. Experiment I — hybrid striped bass and tilapia mortality and bacteriologic eÕaluation after nares and eyes inoculation with S. iniae 3.1.1. Nares inoculation Mortality of hybrid striped bass and the isolation of S. iniae from the moribund and dead fish are shown in Table 1. Maximum mortality was 66.7% at a dose of 4.8 = 10 4

Table 1 Experiment I — Results on hybrid striped bass and tilapia mortality and recovery of S. iniae following nares inoculation Species dose ŽCFU.

No. of morbid fishrtotal fish challenged Ž% mortality.

Mortality onset and peak mortality Ždays.

No. of culture positive fishr morbid fish Ž% fish yielding S. iniae . a

Hybrid striped bass 0b 2r15 Ž13.3. 4.8=10 3 4r15 Ž26.7. 4.8=10 4 10r15 Ž66.7. 4.8=10 5 2r15 Ž13.3. Totals 16r45 Ž35.6.

1, 8 5, 9 2, 7 3, 3

0r2 Ž0.0. 3r4 Ž75.0. 9r10 Ž90.0. 2r2 Ž100. 14r16 Ž87.5.

Tilapia 0b 4.8=10 3 4.8=10 4 4.8=10 5 Totals

– – – 6, 10

– – – 2r3 Ž67.0. 2r3 Ž67.0.

a b

0 Ž0.0. 0 Ž0.0. 0 Ž0.0. 3 Ž20.0. 3 Ž20.0.

Percentage of culture positive fish yielding S. iniae froma Nare Eye Brain

Kidney

– 0 67 50

– 67 33 50

– 67 11 0

– 0 11 50

– – – 100

– – – 100

– – – 100

– – – –

Tissue samples from morbid fish were cultured on blood agar for isolation of S. iniae. Non-infected fish Žcontrols. received tryptic soy broth only by nares inoculation.

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CFU. Mortality decreased to 13.3% following exposure to 4.8 = 10 5 CFU. The majority of hybrid striped bass died within 3 days post inoculation with doses of 4.8 = 10 4 and =10 5 CFU. The lowest dose Ž4.8 = 10 3 CFU. resulted in 26.7% mortality. Mortality onset and peak mortality occurred later in these fish. Most fish given this dose died at 9 days. The majority of morbid hybrid striped bass Ž87.5%. were culture positive for S. iniae ŽTable 1.. Recovery of S. iniae from these morbid fish, irrespective of culture site, increased with increasing doses. S. iniae was recovered from 75%, 90%, and 100% of the morbid fish following inoculation with 4.8 = 10 3 , =10 4 , and =10 5 CFU, respectively. Results of bacteriologic culture of nare, eye, brain, and anterior kidney from culture positive fish were variable. S. iniae was recovered from the eyes and brains of 67% of the culture positive fish inoculated with 4.8 = 10 3 CFU. Likewise, S. iniae was recovered from the nares of 67% of the culture positive fish at that dose causing the greatest mortality Ž4.8 = 10 4 .. S. iniae was isolated from nare, eye, and kidney from at least 50% of the culture positive fish inoculated with 4.8 = 10 5 CFU. Two morbid control fish had heavy gill infection of Ichthyophthirius multifiliis but were absent of S. iniae. Bacteriologic examinations of seven randomly selected control fish and 21 surviving nares inoculated hybrid striped bass sampled at the end of the 14-day study period showed that all the 28 fish were culture negative for S. iniae. Three of 15 tilapia died that had received 4.8 = 10 5 CFU ŽTable 1.. Mortality began on day 6 but ceased at day 12. Doses of 4.8 = 10 3 and =10 4 CFU resulted in no mortalities. Recovery of S. iniae from morbid fish irrespective of culture site identified S. iniae in two of three morbid tilapia Ž66.7%.. S. iniae was recovered from nares, eyes, and brains of these culture positive fish, but not from kidneys ŽTable 1.. Bacteriologic culture revealed two of 28 Ž7.1%. surviving tilapia sampled at the end of the 14-day study period were culture positive at any site. These two tilapia were culture positive by eye and one of these tilapia was also culture positive by nare and brain Ždata not shown.. 3.1.2. Eyes inoculation In contrast to nares inoculation, none of the hybrid striped bass inoculated by eyes died. Four of the four dead control fish had heavy gill infections of I. multifiliis and an absence of S. iniae. Inoculation of the eyes of tilapia also failed to result in S. iniae infection and mortality. No mortality was noted in tilapia controls. Bacteriologic examinations of 20 hybrid striped bass and 28 tilapia sampled at 14-day post eyes inoculation were culture negative for S. iniae. 3.2. Experiment I — serologic response of hybrid striped bass and tilapia after nares or eyes inoculation with S. Iniae 3.2.1. Nares inoculation Results of serologic examination revealed significant anti-streptococcal titers in both hybrid striped bass and tilapia ŽTable 2.. The mean log base10 titers of y1.054, y1.104, and y1.204 were measured in hybrid striped bass receiving doses of 4.8 = 10 3, =10 4 , and =10 5 CFU, respectively. The mean log base10 titer was significantly different between the control fish and those inoculated with these doses. Likewise,

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Table 2 Experiment I — Results of nares and eyes inoculation of S. iniae on antibody titer against S. iniae in surviving hybrid striped bass and tilapia 14 days post inoculation Species

Dose CFU

Mean log base10 titer against S. iniae a by nare inoculation

Mean log base10 titer against S. iniae a by eye inoculation

Hybrid striped bass

0b 4.8=10 3 4.8=10 4 4.8=10 5 0b 4.8=10 3 4.8=10 4 4.8=10 5

y0.376"0.14 A y1.054"0.14 B y1.104"0.20 B y1.204"0.15 B y0.201"0.20 A y0.623"0.28 B y0.753"0.19 B y0.502"0.10 B

y0.376"0.14 A y0.602"0.00 B y0.645"0.10 B y0.129"0.12 A 0.0 A 0.0 A 0.0 A 0.0 A

Tilipia

a Mean log base10 antibody titer"standard error and different superscripts indicate significant differences at P - 0.05. b Non-infected fish Žcontrols. received inoculation of tryptic soy broth only.

tilapia had significant mean log base10 titers of y0.623, y0.753, and y0.502 in fish inoculated with 4.8 = 10 3, =10 4 , and =10 5 CFU, respectively, as compared to control fish Žy0.201.. 3.2.2. Eyes inoculation Anti-streptococcal titers were only detected in hybrid striped bass ŽTable 2.. Serologic responses were significantly different between hybrid striped bass inoculated with 4.8 = 10 3 and =10 4 CFU compared to control fish and those fish inoculated with 4.8 = 10 5 CFU. However, these titers were considerably lower than fish inoculated by their nares. Anti-streptococcal titers were not detected in tilapia following eyes inoculation. 3.3. Experiment II — hybrid striped bass and tilapia mortality and bacteriologic eÕaluation after nares inoculation with S. iniae 3.3.1. Hybrid striped bass Mortality was significant in hybrid striped bass at all S. iniae doses as compared to controls. The results showed 46 of 60 hybrid striped bass Žthree replicates of 20 fish each. died that were inoculated with a dose of 2.6 = 10 4 CFU ŽTable 3.. Fifty five of 60 and 59 of 60 fish died given doses of 5.2 = 10 4 and 1.0 = 10 5 CFU. The mean percent mortality was 76.7, 91.7, and 98.3, respectively. It is important to note here, that mortality in hybrid striped bass inoculated with a higher dose of 4.8 = 10 5 CFU was only 13.3% ŽTable 1.. Total hybrid striped bass mortality over all S. iniae doses was 88.9% in experiment II. The majority of hybrid striped bass died within 3 days post inoculation with doses of 5.2 = 10 4 and 1.0 = 10 5 CFU. Mortality onset and peak mortality occurred later in fish given the lowest dose of 2.6 = 10 4 CFU. The majority of fish died at 6–10 days given

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Table 3 Experiment II — Results on mortality of hybrid striped bass and tilapia infected with S. iniae following nares inoculation Species dose ŽCFU.

No. of fishrtank Ž3 tanksrtreatment.

No. of morbid fishrno. of fish challengeda

Mean% mortality

Mortality onset and peak mortality Ždays. b

Hybrid striped bass 0b 20 2.6=10 4 20 5.2=10 4 20 1.0=10 5 20 Totals

3r60 A 46r60 B 55r60 B 59r60 B 160r180

5.0 76.6 91.7 98.3 88.9

4, 13 2, 7 1, 3 1, 3

Tilapia 0b 1.4=10 7 1.4=10 8 1.4=10 9 Totals

0r60 C 9r60 B 17r60 A 8r60 B 34r180

0.0 15.0 28.3 13.3 18.9

– 2, 9 2, 9 2, 11

20 20 20 20

a Significant differences between mortality among treatment groups are noted by different letters at P - 0.05. b Non-infected fish Žcontrols. received inoculation of tryptic soy broth only.

this dose. Bacteriologic evaluation of the brain and kidney of 23 randomly selected morbid hybrid striped bass revealed that all of these fish were culture positive for S. iniae at 1–2 days post nares inoculation. Three of 60 fish of the control group died of gill and skin infestation of I. multifiliis. Bacteriologic culture of control fish showed the absence of S. iniae at any site of bacteriologic evaluation. 3.3.2. Tilapia Tilapia inoculated with doses of 1.4 = 10 7, =10 8 , and =10 9 CFU showed comparatively lower mortality than hybrid striped bass ŽTable 3.. Seventeen of 60 fish Ž15.0%. died given 1.4 = 10 8 CFU, whereas doses of 1.4 = 10 7 and =10 9 CFU caused mortality of nine of 60 fish Ž28.3%. and eight of 60 fish Ž13.3%., respectively. Like hybrid striped bass mortality, tilapia mortality was significant at all S. iniae doses. Total tilapia mortality over all S. iniae doses was 18.9%. Mortality began at 2 days at all doses. The majority of fish died at 9 days post-inoculation at doses of 1.4 = 10 7 and =10 8 CFU. The majority of fish inoculated with 1.4 = 10 9 CFU died within 11 days. No mortality was observed in the control fish. 3.4. Experiments I and II — hybrid striped bass and tilapia disease signs (behaÕioral and morphological characteristics) after nares or eyes inoculation with S. iniae Disease signs in hybrid striped bass after nares inoculation are shown in Table 4. Moribund hybrid striped bass showed some or all of the following characteristics: swimming at surface or stationary at bottom; erratic, serpentine, rising and falling, and

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Table 4 Experiments I and II — Results of hybrid striped bass and tilapia disease signs Žbehavioral and morphological characteristics. after nares inoculation with S. iniae Species dose ŽCFU.

No. of fish per tankrno. of tanks

Day onset of disease signsa Location Swimming Feeding

Activity

Morphology

Hybrid striped bass 0b 4.8=10 3 2.6=10 4 4.8=10 4 5.2=10 4 1.0=10 5 4.8=10 5

15–20r1–3 15r1 20r3 15r1 20r3 20r3 15r1

– 5 3 3 3 3 3

– 1 2 2 2 2 1

– 2 2 2 2 2 2

– 9 – – 3 3 –

– – – – 2 2 –

Tilapia 0b 4.8=10 3 4.8=10 4 4.8=10 5 1.4=10 7 1.4=10 8 1.4=10 9

15–20, 1–3 15r1 15r1 15r1 20r3 20r3 20r3

– 12 12 9 2 2 2

– 4 2 1 2 2 2

– – 11 7 1 1 5

– 1 – 9 3 3 4

– 1 1 1 4 4 4

a Behavioral characteristics of S. iniae disease were: Ž1. location of fish in the aquarium Žfish surface swimming or stationary at the bottom of the water column.; Ž2. swimming pattern Žfish stationary, fish either swimming on their side, serpentine swimming, head up swimming or frequently sinking and rising.; Ž3. feeding response Žfish slowly accepting or refusing food.; Ž4. activityrexcitability Žfish either hyperactive, lethargic, or unresponsive.. Morphology characteristics of S. iniae disease included darkened skin. b Non-infected fish Žcontrols. received inoculation of tryptic soy broth only.

head up swimming; slow acceptance or refusal of food; lethargy; and dark skin coloration. The moribund fish were first observed to exhibit erratic swimming, darkening of the skin, and slow acceptance of food or refusal of food by 1–2 days post-inoculation. Later, the moribund fish swam close to the surface or were stationary at the bottom of the water column and exhibited lethargic activity by 3 days post-inoculation. The lowest S. iniae dose produced changes in location of fish in aquaria and their activity later Ž5 and 9 days post inoculation, respectively.. Moribund hybrid striped bass did not show exophthalmia, eye opacity, or body curvature. At necropsy, the morbid fish had no petechia, hemorrhages or development of ascites. The control fish had no characteristics associated with S. iniae disease, but the two fish with I. multifiliis infection did show surface swimming and flashing which are characteristics of this disease. Tilapia exhibited disease signs despite no or minimal mortality following nares inoculation ŽTable 4.. Fish initially exhibited all categories of disease signs by 1–4 days post-inoculation following administration of doses G 1.4 = 10 7 CFU. All of these behavioral and morphological characteristics appeared to be influenced by the dose of S. iniae administered. Doses G 1.4 = 10 7 CFU caused earlier onset of disease signs than lower doses. An exception was observed in the onset of dark skin coloration that

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occurred following administration of lower doses at 1 day post-inoculation. Disease signs were observed to occur at the onset of mortality in tilapia at the higher doses and in the absence of mortality at the lower doses. Disease signs of S. iniae infection were largely absent in both hybrid striped bass and tilapia by eyes inoculation. A few fish showed a slight loss of appetite at 1–2 days post-inoculation, but recovered their feeding response by 5–6 days post-inoculation.

4. Discussion Experimental inoculation of hybrid striped bass and tilapia eyes did not result in S. iniae related mortality. Hybrid striped bass and tilapia were susceptible to S. iniae infection following nares inoculation. The mortality rate varied between species by nares inoculation with the same virulent S. iniae isolate and equivalent doses. Based on mortality results, tilapia did not appear to be as susceptible to S. iniae infection as hybrid striped bass by nares inoculation. One possible explanation for the greater susceptibility of hybrid striped bass may be that the source of the S. iniae isolate was from hybrid striped bass and this isolate had greater virulence for hybrid striped bass than tilapia. In tilapia, higher doses were required to initiate mortality but mortality levels did not approach those of hybrid striped bass at any dose. In addition, we found that mortality decreased in hybrid striped bass nares inoculated with 4.8 = 10 5 CFU and in tilapia inoculated with 1.4 = 10 9 CFU. The reason for the decreases in fish mortality in both species following nares inoculation with a higher concentration of S. iniae organisms than necessary to initiate infection and mortality are unknown, but warrants further investigation. The bacteriologic analysis showed that S. iniae was isolated from the majority of morbid hybrid striped bass Ž87.5%. and tilapia Ž66.7%. indicating that these fish were successfully infected with S. iniae. The treatment groups, which experienced the greatest mortality, had the greatest percentage of morbid fish culture positive by the nares. Sixty-seven percent of culture positive morbid hybrid striped bass and 100% of culture positive tilapia yielded S. iniae at the nares site suggesting a relationship between nare colonization and S. iniae infection. Conversely, the inabiltiy to recover S. iniae from nares or other culture sites from either species of seemingly healthy surviving fish at the end of the 14-day study period suggests that the lack of organ colonization may be related to survival. There are few documented cases of S iniae infection in hybrid striped bass. Perera et al. Ž1997. reported disease and mortality in this species following experimental intraperitoneal injections with a biotype of S. iniae Žthe Texas isolate. but few fish were used and mortality rates were not given. They observed less pronounced external disease signs which consisted of petechiae around the mouth and anus in hybrid striped bass than in tilapia. Stoffregen et al. Ž1996., in a report of an outbreak of S. iniae in cultured hybrid striped bass, observed hemorrhage and ulceration of the skin and ocular lesions Žcorneal opacity, hemoptyon, and hypopyon. in moribund fish but did not report on behavioral anomalies. We did not observe any of the previously reported skin or ocular lesions in hybrid striped bass nares inoculated.

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Disease signs have been reported for S. iniae infections of cultured tilapia ŽBowser et al., 1998; Perera et al., 1998. and experimentally injected tilapia ŽPerera et al., 1994, 1997.. Dermal hemorrhages on the body surface ŽBowser et al., 1998. and around the mouth, opercles, base of fins and anus ŽPerera et al., 1994. have been observed in S. iniae infected moribund tilapia. Perera et al. Ž1994. also reported loss of orientation, corneal opacity, exophthalmia, and eye disfigurement. In nares inoculated tilapia, we did not observe the skin and eye lesions previously reported but rather we observed darkened skin pigment, erratic surface, or bottom swimming that included serpentine, rising and falling, and side swimming and slow acceptance or refusal of food. Our findings indicate that the onset of disease signs in hybrid striped bass and tilapia occurred simultaneously with the initiation of mortality. Disease signs also occurred later in the absence of mortality in both species at the lower doses. We did not observe disease signs of S. iniae in either hybrid striped bass and tilapia following eyes inoculation. Many observations of disease signs, both behavioral and gross anatomical, have been made on fish infected with uncharacterized Streptococcus sp. Often times, these observations are assumed to be identical to those that occur from S. iniae infections. In addition, differing disease signs may vary according to natural and artificial S. iniae infections and between experimental routes of infection. Furthermore, disease signs reported in the literature from either streptococcal species or S. iniae are often confounded by a clear presentation of which pathogen is responsible. In the published literature that described disease signs and mortality due to S. iniae, regardless of the route of infection, data on the disease signs, mortality and isolation of S. iniae from morbid fish are not well defined. Given the aberrant fish behavior, darkened skin and isolation of S. iniae from the central nervous system of hybrid striped bass and tilapia in this study, it would appear that S. iniae is neurotropic. Histological findings from naturally infected morbid hybrid striped bass and tilapia have included severe, extensive meningoencephalitis and uveitis associated with a large number of intracellular cocci, presumably S. iniae ŽPerera et al., 1998; Stoffregen et al., 1996.. We established that hybrid striped bass and tilapia produced anti-streptococcal antibodies by nares inoculation. Only hybrid striped bass produced anti-streptococcal antibodies by eyes inoculation. The finding that S. iniae was capable of stimulating antibody by eyes inoculation was surprising since we were unable to observe mortality or disease signs following eyes inoculation. It is possible that S. iniae was able to stimulate an antibody response but not fatal infection due to its survival in eye tissues. This is a noteworthy result that requires further investigation. Furthermore, infectivity of the fish may be related to length of exposure of the eye to S. iniae, dose of S. iniae or factors other than inoculation protocols such as anatomy, physiology and appropriate mucosal receptors in the eye. In addition, the eye may be the end result of chronic S. iniae infections, not the initial starting point of the disease process. The pathology of S. iniae from the eyes of naturally infected hybrid striped bass and tilapia may occur from the internal invasion of S. iniae from the olfactory organ rather than direct invasion of the eye tissue. Likewise, the brain may become infected from the dissemination of S. iniae from the olfactory organ. In pathogenesis studies of enteric septicemia of channel catfish, Edwardsiell ictaluri, was shown to cause disease signs, mortality, and brain

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pathology following experimental nares inoculation ŽShotts et al., 1986; Morrison and Plumb, 1994.. The natural pathology of the disease or pathogenesis has not been defined for S. iniae. Our work suggests that the olfactory organ is an experimental site of S. iniae infection for hybrid striped bass and tilapia. Mortality, isolation of S. iniae from morbid fish, disease signs, and serological responses indicated successful S. iniae infection following nares inoculation. Further experimentation will be necessary to firmly establish the importance of the nares in S. iniae infections and pathogenesis. The authors would like to thank Paige Hendrix and Ning Qin for their excellent technical assistance and Dr. John Plumb and Dr. G. Reed Bishop for their helpful comments in the preparation of this manuscript.

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