Identification and Epidemiology of Streptoccocus iniae and

S. agalactiae

Joyce J. Evans, Phillip H. Klesius, Craig A. Shoemaker and David J. Pasnik

USDA/ARS

Aquatic Animal Health Research Laboratory

Chestertown, MD and Auburn, AL

Streptococcus iniae and S. agalactiaeStreptococcus iniae and S. agalactiae Overview

• Global Distribution• Clinical Disease Signs• Fish host susceptibility & alternative hosts• Sample Collection, Transport & Storage• Characterization

– Conventional , automated & molecular diagnostic techniques

• Environmental Influences or parasitism on disease susceptibility

Streptococcus iniae and S. agalactiaeStreptococcus iniae and S. agalactiae

• Highly fatal bacterial fish pathogens implicated also in human & bovine infections

• Major disease problem in food fish production and feral fish worldwide

• Neurotropic disease with high mortality

• More than 40 species of fish affected

• Affects fish from diverse habitats

Estuarine & Marine fish species affected by S. iniae & S. agalactiae

Bottlenose dolphin (Tursiops truncatus)*• Gulf menhaden (Brevoortia patronus)*• Spot (Leiostomus xanthurus)*• Atlantic croaker (Micropogon undulatus)*• Sea catfish (Arius felix)*• Striped mullet (Mugil cephalus)*• Pinfish (Lagodon rhomboids)*• Stingray (Dasyatis sp)*• Silver trout (Cynoscion nothus)*• Sea trout (Cynoscion regalis)*• Gulf killifish (Fundulus grandis)*• Bluefish (Pomatomus saltatrix)*• Klunzingeri mullet (Liza klunzingeri)*• Gilthead sea bream (Sparus auratus)*• European sea bass (Dicentrarchus labrax)• Silver pomfret (Pampus argenteus)• Barramundi (Lates calcarifer)

• Yellowtail (Seriola quinqueradita) • Snapper (Ocyurus chrysurus)• Grunt (Haemulidae sp)• Brown spot grouper (Epinephelus

coioides)• Borneo grouper (Liza macrolepis)• Lined piggy (Pomadasys stridens)• Parrot fish (Sparisoma aurofrenatum)/

(Sparisoma viride)• Black margate (Anisotremus sp)• Chubb (Scaridae sp.)• Common mackerel (Scomber japonicus)• Japanese/ Olive flounder (Paralicthys

olivaceus)• Whitespotted spinefoot “ rabbitfish”

(Siganus canaliculatus)• Marbled spinefoot (Siganus rivulatus)• Dusky spinefoot (Siganus fuscescens)• Lizard fish (Synodus variegates)

Freshwater fish species affected by S. iniae & S. agalactiae • Amazon dolphin (Inia

geoffrensis)• Mossambique tilapia

(Oreochromis mossambicus)• Blue tilapia (Oreochromis

aureus)• Nile tilapia (Oreochromis

niloticus)*• Tilapia hybrid (Oreochromis

niloticus x O. mossambicus)*• Red tilapia (Oreochromis

niloticus x O. aureus)*• Tilapia spp unspecified

(Oreochromis spp.)*

• Golden shiners (Notemigonus crysoleucas)

• Rainbow trout (Oncorhynchus mykiss)

• Coho salmon (Oncorhynchus kisutch)

• Amago (Oncorhynchus rhodurus var. macrostomus)

• Ayu (Plecoglossus altivelis)

Gray mullet (Mugil cephalus)• Hybrid striped bass/ Sunshine

bass (Morone chrysops x M. saxatilis)

• Striped bass (M. saxatilis)

Streptococcus iniae & S. agalactiae in tilapia

Countries where Streptococcus iniae has been reported ( 9 )Countries where Streptococcus agalactiae has been reported ( 3 )Countries where both Streptococcus iniae and Streptococcus agalactiae have been reported (4 countries, 3 continents)

S. iniae & S. agalactiae in tilapia species

Oreochromis niloticus

F / N & E Japan, U.S.A.F / N Thailand, Indonesia, Venezuela, Ecuador, Honduras

F / N Thailand Brazil, U.S.A (E) SA

Oreochromis mossambicus  M / N Israel

F / N Taiwan

Oreochromis aureusF / E U.S.A.

 Oreochromis niloticus x O. aureusF / E U.S.A.

  F / N Saudi Arabia, IsraelSA

Oreochromis spp.F / N Israel, U.S.A, Canada, Columbia, Philippines, China

Oreochromis niloticus x O. mossambicus F / N ThailandSA

S. iniae and S. agalactiae Clinical & Behavioral Signs of Disease

are similar

• Erratic swimming behavior • “C-shaped” body posture• Exophthalmia, multiple ocular

lesions • Lethargy, Off feed• Fecal cast & operculum clear-S.

agalactiae

S. iniae/ S. agalactiae experimental infections Nile tilapia behavioral signs “C” shape/ erratic swimming

Head up Upside down/side to side

Head down

Nile tilapia S. iniae/ S. agalactiae disease signs

Ocular and opercula SA lesions

missing eye

opaque eye

cloudy eye

collapsed eye

hemorrhagic eye

Popeye

Body and fin hemorrhagic lesions

Streptococcus iniae & S. agalactiae

IsolationMicrobiological Collection, Transport & Storage

• Culture sites-lethal (brain, HK)/ non-lethal (Nares)– Evans et al., 2000,2001

• Transport system for maintaining viability– Evans et al., 2002

• Survival in frozen fish(archival, retrospective)– Evans et al., 2004

Genus Identification of catalase negative Gram positive cocci in chainsBasis of confusion

  Genera Hemolysis Lancefield Starch Growth

Hydolysis @10 45 6.5% NaCl

 

Streptococcus α/β/n A-H, L, K-N, +/- v  v  -  O,U,V, ng

Enterococcus α/β/n  D  +/-  +  +  + Lactococcus   α/n  N   -  +  -c  v

Conventional tests to confirm Streptococcus genus and/or species

• Hemolysis on 5% SBA• Motility

• Growth @10°C45°C

in 6.5% NaClbroth • Reaction on Bile-Esculinmedia • Production of:

Pyrroliydonyl arylamidase PYR Leucine aminopeptidase LAP • Gas from glucose in MRS broth • Susceptibility to Vancomycin (30 ug)• Vogas-Prokauer (VP) reaction• CAMP test

Si Sa+ +/-- -+ -- -- -- -

+ -+ +- nr+ +- -+ +

Conventional techniques to differentiate between S. iniae & S.agalactiae

S. iniae• No Lancefield

group –non groupable

• Starch hydrolysis +

S. agalactiae• Lancefield

group B• Starch

hydrolysis –

Commercial multi-test systems & Molecular techniques

S. iniae• API rapid strep 32- No

S.iniae ID-Not in database• Biolog- excellent ID

• Monoclonal antibody-based indirect fluorescent antibody technique-

Klesius et al., 2006

S. agalactiae• API rapid strep 32-

S. agalactiae ID• Biolog- excellent ID

Streptococcus iniae researchPathogenesis • S. iniae infectivity and distribution after eye, nare &

gill inoculation in Nile tilapia Evans et al., 2000;2001; McNulty et al., 2003

• Effect of density on infection- densities of 11.2 g/L and above sig effect on S. iniae mortality Shoemaker et al, 2000

• Amoxicillin oral feed treatment prior to and during S. iniae infection in Nile tilapiano sig differences in mortality-Evans unpublished

Streptococcus agalactiae researchStress & Disease Susceptibility in Nile tilapia• Environmental influences (DO, UIA) reported as key components in epizootics• Sublethal D0 stress- Evans et al., 2003

• Sublethal Unionized

ammonia- (0.35 mg/L)

Evans et al., 2006

• Increased susceptibility to S. agalactiae at elevated salinities (30 ppt) at 20 and 30°C Chang and Plumb, 1996

1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 140

5

10

15

20

25

0

1

2

3

4

5

6

7

8

9

10

Days

S. Iniae & S. agalactiae research

Disease Susceptibility in non-traditional parasitized & non parasitized host-Channel catfish

Reduced survival among Trichodina sp parasitized channel catfish fry challenged with either S. iniae or S. agalactiae Evans et al in press

Challenged, parasitized fish: Challenged, non-parasitized fish:• Clinical signs No clinical signs• Streptococcus sp. cultured No Streptococcus sp. cultured• 0 to 73% survival 88 to 100% survival

Streptococcus iniae and S. agalactiae Vaccine development

Early indicators of needU.S. wide epidemiology survey of the incidence of Streptococcus iniae in catfish, tilapia and hybrid striped bass production farms indicated prevalence

Shoemaker, Klesius, Evans, 2001

Worldwide epizootics

Fish epizootics in Gulf of Mexico (Plumb et al 1974), Chesapeake Bay (Baya et al 1990) & Kuwait Bay (Evans et al., 2002; Glibert et al., 2002), Thailand (Suanyuk et al 2005) and Brazil (Salvadore et al 2005)

due to S. agalactiae.

Acknowledgments

• Dr. Richard Shelby- USDA/ARS, Auburn, AL• Crystal Braden- USDA/ARS, Auburn, AL• Daniel Brougher- USDA/ARS, Chestertown, MD• Lisa Biggar- USDA/ARS, Chestertown, MD