Identification and Epidemiology of Streptoccocus iniae and S. agalactiae

1
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

2
Streptococcus 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

3
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

4
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)

5
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)

6
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)
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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 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

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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

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S. iniae/ S. agalactiae experimental infections
Nile tilapia behavioral signs C shape/ erratic
swimming
Upside down/side to side
Head up
Head down
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Nile tilapia S. iniae/ S. agalactiae disease signs
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Ocular and opercula SA lesions
cloudy eye
hemorrhagic eye
opaque eye
Popeye
missing eye
collapsed eye
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Body and fin hemorrhagic lesions
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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

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

•  
• Genera Hemolysis Lancefield Starch
  Growth
• Hydolysis _at_10? 45? 6.5
  NaCl
•  
• Streptococcus a/ß/n A-H, L, K-N, /-
  v  v  -
•   O,U,V, ng
• Enterococcus a/ß/n  D 
  /-     
•  
• Lactococcus   a/n  N
    -    -c  v

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Conventional tests to confirm Streptococcus genus
and/or species

• Si Sa
• /-
• - -
• -
• - -
• - -
• - -
• -
• - nr
• - -

• Hemolysis on 5 SBA
• Motility
• Growth _at_10C
• 45C
• 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

16
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

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Commercial multi-test systems Molecular
techniques

• S. agalactiae
• API rapid strep 32- S. agalactiae ID
• Biolog- excellent ID

• 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

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Streptococcus iniae research

• Pathogenesis
• S. iniae infectivity and distribution after eye,
  nare gill inoculation in Nile tilapia
• Evans et al., 20002001 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

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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
• Sublethal Unionized
• ammonia- (0.35 mg/L)
• Evans et al., 2006
• Increased susceptibility to S. agalactiae at
  elevated salinities (30 ppt) at 20 and 30C
  Chang and Plumb, 1996

20
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

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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.

22
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