http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/ FAO Second International Technical Seminar/Workshop on Acute hepatopancreatic necrosis disease (AHPND) There is a way forward! FAO Technical Cooperation Programme: TCP/INT/3501 and TCP/INT/3502.

1. ECOLOGY, VIRULENCE FACTORS
AND GLOBAL SPREAD OF
PATHOGENIC VIBRIO
PARAHAEMOLYTICUS AND
RELATED VIBRIO SPP
Iddya Karunasagar
Iddya.Karunasagar@gmail.com

2. Virulence factors of AHPND causing strains
• A 69kbp plasmid (pVA1, pVPA3-1) found in V. parahaemolyticus
causing AHPND (Lee et al., 2015; Han et al., 2015).
• Two proteins with 33% identity to PirA and PirB toxins shown to be
essential to cause characteristic pathology.
• Five transposase genes were found on this plasmid
• The plasmid has a cluster of genes involved in conjugal transfer of
plasmid

3. Virulence factors of AHPND causing strains
• On plasmid pVPA3-1, the two Pir genes are located within a 3.5 kb-
long insertion element (IS) flanked with inverted repeats.
• these inverted repeats are from an insertion element of IS903 family.
Mobile/transposable elements (TEs) such as IS5 family are involved in
the spread of pathogenicity via horizontal gene transfer (HGT).

4. Virulence factors of AHPND causing strains may be present in
other Vibrio spp.
• These suggest potential for mobility of plasmid and gene clusters within the
plasmid.
• Strain 13.17.5 isolated from AHPND affected shrimp in Vietnam carried virulence
plasmid. This strain has been sequenced and based on 16S rRNA sequence, has
been identified as V. harveyi (Kondo et al., 2015).
• V. owensii strain SH14 isolated from AHPND affected shrimp in China carried the
69kb plasmid with virulence genes (Liu et al., 2015).
• Hepatopancreatitis reported from Australia did not meet the case definition of
AHPND. Member of V. harveyi clade carrying PirAB genes, but not other plasmid
markers has been isolated.
• Can virulence gene cluster integrate into chromosome?

5. Genetic diversity of AHPND causing strains
• Multi locus sequence typing (MLST) of AHPND strains from Thailand show that
they are genetically diverse (Chonsin et al., 2016)
• Loss of virulence gene in plasmid carrying strains reported (Lee et al., 2015).
• V. harveyi clade lacking plasmid markers, but positive for PirAB toxin genes
reported from Australia.

6. Are the toxin genes found in AHPND causing V.
parahaemolyticus new to marine environment?
• The binary toxin gene of AHPND V. parahaemolyticus has 33% identity with
PirAB gene
• These toxins have 40% -44% identity to two hypothetical proteins of
Shewanella violacea!
• Shiwanella violacea was isolated from marine trench at a depth of 5110m.
It is an obligate psychrotroph.
• Thus the toxin genes may not be new to aquatic and marine environment.
• These PirAB genes have a G+C content of 38.2%, substantially lower than
45.9% in the plasmid. This suggests that this gene cluster is recently
acquired by the plasmid (Han et al., 2015).

8. AHPND virulence plasmid
• The pVA1 plasmid also carries the pndA gene, which is associated with a post-
segregational killing (psk) system.
• With a bacterium that harbours a plasmid with the psk system (PSK+), only
progeny that inherit the PSK+ plasmid will be viable.
• Progeny that do not inherit the PSK+ plasmid will die because the stable pndA
mRNA will be translated to PndA toxin that will kill the bacterium.
• The presence of a psk system on a plasmid thus ensures that the plasmid is
inherited during bacterial replication.
• The pVA1 plasmid will therefore be passed on to subsequent generations of VP-
AHPND producing PirAB toxin.

9. Is the Mexican AHPND strain different from Asian strain?
• Clear genetic variation exists between Asian and Latin American strains
(Han et al., 2015).
• A 4243bp TN3 like transposon element found in Mexican and Central
American strains, but not in strains from Vietnam, Thailand and China.
• The number of 9bp small sequence repeats (SSR) found within the coding
region of a hypothetical protein vary in Asian and Latin American strains.
• SSR with 6 repeats found in Mexican strains, 5 repeats in Vietnamese
strains and 4 repeats in China, Thailand and Vietnamese strains.
• PCR based on SSRs could differentiate Mexican type and SE Asian type
strains.

10. Molluscs
V. cholerae
V. parahaemolyticus
V. vulnificus
V. cholerae
V. parahaemolyticus
V. vulnificus
Modified from Reen et al, 2006

11. V. parahaemolyticus – occurrence and distribution
• Estuarine and coastal environments
• Global occurrence
• Associated with all types of animals in brackish water environments –
zooplankton, molluscs, crustaceans, shellfish and finfish
• Ecology influenced by temperature and salinity.
• In tropical environments, detected throughout the year, provided salinity and
other conditions are available.
• In temperate climate, detected mostly in summer. The organism undergoes
overwintering in sediments.

12. V. parahaemolyticus – occurrence and distribution
• During the 1990’s, the organism was considered mainly tropical.
• In international fish trade, there were even rejections due to the mere presence
of the organism (though most environmental organisms are non-pathogenic to
humans)
• An outbreak of V. parahaemolyticus gastroenteritis in Alaska in 2004 from locally
grown oysters changed the thinking on the distribution of this organism.
• Alaska is 1000KM north of places from where any previous V. parahaemolyticus
illness was reported.
• In 2004, temperatures in Alaska were higher that 15°C.

13. V. parahaemolyticus – occurrence and distribution

14. Metabolic versatality of Vibrio spp
• Capable of both oxidative and fermentative metabolism.
• Quick reproduction (15-20 min), high content of tRNA genes (126 in V.
parahaemolyticus) compared to bacteria in general (around 50 genes).
• tRNA genes also hotspots for insertion of novel DNA and gene acquisition.
• Presence of two chromosomes, larger chromosome carries most essential genes
and the smaller chromosome carries species specific genes.
• Quorum sensing adaptive mechanism to stress such as low nutrient, exposure to
UV, predation, salinity fluctuation. In V. parahaemolyticus, the regulatory protein
is LuxO.

15. Metabolic versatality of Vibrio spp
• Ability to form biofilms for survival in different environments. Syp gene cluster in
V. parahaemolyticus.
• Survival of acid stress mediated by proteins encoded by cadBAC and heat stress
mediated by GroEL heat shock protein.
• Production of proteins to enable the organism to tolerate cold stress, osmotic
stress.
• Ability to bind to and degrade chitin.

16. Interaction between V. parahaemolyticus and molluscs and
crustacea
• When temperatures are favourable, V. parahaemolyticus can grow in American
oysters Crassostrea virginica.
• No growth is observed in Sydney rock oysters stored at 30°C for seven days.
• Studies done in Mangalore, India indicate presence of V. parahaemolyticus in
oysters at levels ranging from 102 to 104 per gram throughout the year.
• Can be detected in shrimp farms in India throughout the year accounting for 3-
10% of the flora (Gopal et al., 2005)

17. Interaction between V. parahaemolyticus and plankton
• V. parahaemolyticus can adhere to and degrade chitin and adsorption of the
organism to chitin or plankton occurs with higher efficiency under conditions of
low estuarine salinity.
• Adherence to chitin could explain the relation between abundance of
zooplankton and presence of this organism.
• In a study in Galacia, Spain (Martinez-Urtaza et al., 2011), 80% of V.
parahaemolyticus biomass was associated with zooplankton. The presence of
cnidarians accounted for 52% of variation in abundance, though they constituted
only 2% of zooplankton.
• Chlorophyll-A may influence the ecology of the organism by influencing the
population of zooplankton.
• In finfish, higher prevalence is observed in the gut compared to gills and surface.

18. Role of plankton in oceanic spread of V. parahaemolyticus
• Aquatic birds may be carriers of V. parahaemolyticus. Studies in Japan and other
countries show the presence of this organism in feces of aquatic birds, though the
concentrations could be low.
• Studies done in Galecia, Spain indicate that V. parahaemolyticus is found even in
off-shore areas in association with zooplankton.
• Genetically similar organisms were found in zooplankton in estuaries and off-
shore regions spread over 1500 KM suggesting a role for zooplankton in the
spread of this organism.

19. Global spread of pandemic strain of V. parahaemolyticus

20. Global spread of pathogenic strains
• Ballast water?
• Seafood transport?
• Some insights into the global spread of V. parahaemolyticus has been provided by
studies performed on the strains that caused outbreaks of infection in Chile in
2004.
• The pandemic strain belonging to O3:K6 serovar arrived in Peru in 1997 and
infections were reported in northern part of the country, but spread southward
along more than 1500 KM of the coast till it reached the Chilean city of
Antofagasta.

21. Global spread of pathogenic strains
• Molecular studies of the strains suggest that the 1997 El Nino episode provided
the corridor for the displacement of the Asian pandemic V. parahaemolyticus
strains to America.
• The El Nino phenomenon is characterized by the arrival of equatorial warm
waters in a sequence of waves. In 1997, El Nino affected South American coast for
about 6 months.
• It has been suggested that recurrent invasion of tropical masses of water might
have resulted in repetitive sources of V. parahaemolyticus populations that would
have established there.

25. Molecular genetic studies show the sequence types that have spread with the El Nino
event
Gonzales-Escalona et al., 2015

26. Outbreak of V. parahaemolyticus infection in Spain involving
strains previously associated with outbreaks in US
• Martinez-Urtaza et al. (2016), investigated strains of V. parahaemolyticus involved
in an outbreak that affected 100 of 114 passengers travelling in a food banquet
cruise in Galacia, Spain in 2012.
• Strains were positive for both tdh and trh genes (usually they are positive for one
of them) and were never detected in Europe before.
• Molecular typing using PFGE, MLST showed that the strains are highly related to
those associated with human cases in Pacific North West of United States.
• This supports theory of oceanic spread, though the route of spread has not yet
been established.

27. Questions with respect of AHPND causing strains
• Ecology and distribution, association with aquatic animals, zooplankton.
• The environmental source of the plasmid found in these strains.
• Can the plasmid be integrated into chromosome? What are the implications for
virulence?
• Can the plasmid be transferred to human pathogenic strains of V.
parahaemolyticus?
• Did the Mexican strain originate in Mexico or is it derived from Asian strains?
• Are other Vibrio spp carrying the virulence plasmid equally virulent?

28. Is the Mexican AHPND strain different from Asian strain?
• Clear genetic variation exists between Asian and Latin American strains (Han et
al., 2015).
• A 4243bp TN3 like transposon element found in Mexican and Central American
strains, but not in strains from Vietnam, Thailand and China.
• The number of 9bp small sequence repeats (SSR) found within the coding region
of a hypothetical protein vary in Asian and Latin American strains.
• SSR with 6 repeats found in Mexican strains, 5 repeats in Vietnamese strains and
4 repeats in China, Thailand and Vietnamese strains.
• PCR based on SSRs could differentiate Mexican type and SE Asian type strains.
6/25/2016
International Technical Seminar/Workshop “EMS/AHPND:
Government, Scientist and Farmer Responses”
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29. Summary
• Virulence genes associated with AHPND V. parahaemolyticus may be found in
other Vibrio spp such as V. harveyi and V. owensii.
• Genetically diverse strains of Vibro spp may carry the virulence plasmid.
• Vibrio parahaemolyticus is a very versatile organism adapting to a number of
environments.
• The organism is capable of acquiring gene clusters to adapt to different hosts.
• Association of the organism with zooplakton facilitates its global spread through
ocean currents like El Nino phenomenon.
• It is also possible that Latin American strains emerged independent of Asian
strains.

30. 6/25/2016
International Technical Seminar/Workshop “EMS/AHPND: Government, Scientist and Farmer
Responses”
30
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