Transcript Identification and Epidemiology of Streptoccocus iniae and S. agalactiae Joyce J. Evans, Phillip H.

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. agalactiae
Overview
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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. 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
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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)
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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.)*
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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
F/N
Japan, U.S.A.
Thailand, Indonesia, Venezuela, Ecuador, Honduras
F/N
Thailand Brazil, U.S.A (E) SA
Oreochromis mossambicus
M/N
F/N
Israel
Taiwan
Oreochromis aureus
F/E
U.S.A.
Oreochromis niloticus x O. aureus
F/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
hemorrhagic eye
Popeye
collapsed eye
cloudy eye
opaque eye
missing eye
Body and fin hemorrhagic lesions
Streptococcus iniae & S. agalactiae
Isolation
Microbiological 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 chains
Basis of confusion
Genera Hemolysis Lancefield Starch
Hydolysis
Streptococcus α/β/n
Enterococcus α/β/n
Lactococcus
α/n
A-H, L, K-N,
O,U,V, ng
Growth
@10 45 6.5%
NaCl
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N
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v
Conventional tests to confirm
Streptococcus genus and/or species
• Hemolysis on 5% SBA
• Motility
• Growth @10°C
45°C
in 6.5% NaCl broth
• Reaction on Bile-Esculin media
• 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
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Sa
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Conventional techniques to differentiate
between S. iniae & S.agalactiae
S. iniae
S. agalactiae
• No Lancefield
group –non
groupable
• Starch hydrolysis
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• Lancefield
group B
• Starch
hydrolysis –
Commercial multi-test systems &
Molecular techniques
S. iniae
S. agalactiae
• API rapid strep 32- No
S.iniae ID-Not in database
• Biolog- excellent ID
• API rapid strep 32S. agalactiae ID
• Biolog- excellent ID
• Monoclonal antibody-based
indirect fluorescent
antibody techniqueKlesius et al., 2006
Streptococcus iniae research
Pathogenesis
• 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 tilapia
no sig differences in mortality-Evans unpublished
Streptococcus agalactiae research
Stress & Disease Susceptibility in Nile tilapia
• Environmental influences (DO, UIA)
reported as key components in epizootics
• Sublethal D0 stress- Evans et al., 2003
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ammonia- (0.35 mg/L)
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Evans et al., 2006
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Days
• Increased susceptibility to S. agalactiae at elevated salinities
(30 ppt) at 20 and 30°C Chang and Plumb, 1996
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:
• Clinical signs
• Streptococcus sp. cultured
• 0 to 73% survival
Challenged, non-parasitized fish:
No clinical signs
No Streptococcus sp. cultured
88 to 100% survival
Streptococcus iniae and S. agalactiae
Vaccine development
Early indicators of need
U.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
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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