prevalence and molecular characteristics of vibrio species in pre ...

2.1 Shellfish culture
CHAPTER II
LITERATURE REVIEW
Shellfish culture plays an important role in the world’s aquaculture industry.
It has continued to expand with ever increasing consumer demands. Shrimps, oysters,
mussels and crabs are some species included in shellfish cultures. Among them
shrimp is the most popular in the aquaculture industry.
Shrimp belongs to the largest phylum in the animal kingdom and are
Arthropod and Decapods crustaceans of suborder Natania. They are characterized by
joined appendages and a periodically moulted exoskeleton (see Figure 1). The term
“shrimp” and “prawn” are used interchangeably throughout the world for different
species. According to FAO convention “shrimp” is used for marine or brackish water
forms and “prawn” is for fresh water forms (FAO, 2008).
Figure 1: Anatomy of the shrimp.
(http://www.shrimpnews.com/AnatomyShrimp.html)

2.2 Shrimp production and trade
6
The shrimp industry has boomed during last two decades. South America and
Asia are the two predominant areas of larger scale shrimp production in the world.
South Asian countries contribute 80% of the total world production. Even though
there are many shrimp species, only two of them dominate the industry.
1. Black tiger shrimp (Penaeus monodon) - widely spread in Asian countries,
such as Thailand, Indonesia, India, Vietnam, Sri Lanka, Philippines and
Malaysia
2. Pacific white shrimp (Penaeus vannamei) - commonly found along the Eastern
coast of the Pacific Ocean countries such as Brazil, Ecuador, Panama, Peru
and Mexico.
Currently, the white shrimp (Penaeus vannamei) is rapidly replacing the black
tiger shrimp as the main farmed species in Asia. This transformation started in Taipei
China in the late 1990s with the importation of specific-pathogen-free brood stock of
white shrimp from Hawaii and is now cultured in large scale in the People’s Republic
of China,
Thailand, Indonesia and Vietnam. The reasons for this change is that Penaeus (P.)
vannamei has a faster growth, high yield, low production cost and is able to survive
with a high stocking density when compared with black tiger shrimp (Chinabut et al.,
2006). White shrimps grow to a maximum commercial size of 20g while black tiger
shrimp grow up to 30g. The disadvantage of P. vannamei is the low market price in
the international trade when compared with black tiger shrimp. Therefore large scale
production is necessary in order to generate profits (Weerakoon, 2007).
Over the last decade, the world shrimp production increased remarkably. Data
from the Food and Agriculture Organization (FAO) is shown in Table 1, the total
cultured and wild harvest shrimp production in the year 2000 was 4249 thousand
tones and it increased up to 6624 thousand tonnes in the year 2006. Even though wild
shrimp production is seasonal and fluctuates, production remains more or less

7
constant over the years while cultured shrimp production has steadily increased.
Cultured shrimp production represented 27.3% of the total cultured and captured
production in the year 2000 and it increased by almost half (48.0%) by the year 2006
(Wan Norhana et al., 2010).
Thailand, Indonesia, India, Vietnam, Sri Lanka, the Philippines and Malaysia
contribute about 60% of the world’s total cultured shrimp production (Chinabut et al.,
2006). The demand for shrimp in world trade has also increased steadily during the
last decade. According to the FAO reports, in 1986, world shrimp exports totalled
about 1.4 million metric tonnes and it tripled to almost 4.5 million metric tonnes by
2006 (Table 2) (Wan Norhana et al., 2010).
Table 1: World shrimp production from 2000 to 2006 (FAO, 2009)
Source Value 2000 2001 2002 2003 2004 2005 2006
Capture 1000
tonnes
US$
million
Aquaculture 1000
tonnes
US$
million
Total 1000
tonnes
US$
million
3087 2955 2966 3543 3527 3420 3460
11,175 10,411 9788 11,621 11,357 11,458 11,764
1162 1347 1496 2129 2446 2716 3164
7310 7492 7879 8355 9536 10,501 12,486
4249 4302 4462 5672 5973 6136 6624
18,458 17,893 17,667 19,976 20,893 21,959 24250

Table 2: World exports of shrimp
International export of shrimp by FAO ISSCAAP (International Standard Statistical
Classification of Aquatic Animal and Plant)
Value 1986 1996 2006
Tonnes 938,102 1,601,147 3,244,871
US$ 1000 4,740,879 9,957,324 14,138,751
2.3 Shrimp consumption
8
Shrimp is considered a luxury food commodity because of its unique texture
and flavour. According to reports (Bragagnolo and Rodriguez-Amaya, 2001), the high
cholesterol content of shrimp is compensated by the very low total lipid content and
the predominance of poly unsaturated fatty acids, especially the omega 3 fatty acid,
which is important for human health. Another advantage of shrimp meat is the low
mercury content when compared with other seafood. Consumer demand for shrimp is
increasing in most developed countries. According to the reports of the United States
National Marine Fisheries Service (NMFS) shrimp consumption per capita has
increased over the years from an average of 1kg per year in 1989 to a record high of
1.8kg in 2005. Similarly, the European Union and Australia show a high consumer
demand for the shrimp (Wan Norhana et al., 2010).
2. 4 Importance of pathogenic bacteria in shrimp production
The presence of pathogenic bacteria in shrimp is closely related with
environmental conditions and the microbiological quality of the water. Water
temperature, salt content, distance between localization of catch and polluted areas,
natural occurrence of bacteria in water, ingestion of food by the shrimp, method of
catch and chilling conditions determine the number and the type of bacteria in the
shrimp (Feldhusen, 2000).

9
Pathogenic bacteria associated with seafood can be categorized in to three groups
(Feldhusen, 2000). These are
Indigenous bacteria: Normal component of the marine and estuarine
environment.
Eg: V. cholerae, V. parahaemolyticus, V. vulnificus, Listeria monocytogenes,
Clostridium botulinum, Aeromonas hydrophila (only virulent strains)
Enteric bacteria: Due to contamination by the fecal material of animals and
human
Eg: Salmonella spp., pathogenic Escherichia coli, Shigella spp.,
Campylobacter spp., Yersinia enterocolitica (very few pathogenic sero types)
Bacterial contamination during processing: (cross contamination)
Bacillus cereus (only toxigenic strains), L. monocytogenes, Staphylococcus
aureus, Clostridium perfringens.
Although many pathogenic bacteria are associated with the shrimp production
chain only a few organisms (Salmonella, Vibrio and Listeria) have been thoroughly
studied for their prevalence and public health importance (Wan Norhana et al., 2010).
Bhaskar et al. (1998) found Salmonella and Vibrio spp. in all the samples of shrimp,
sediment, feed and pond water during the farming phase and at harvesting time.
Listeria spp. was found only in clam meat during farming and sediment and shrimp at
harvest. A study done in Sri Lanka revealed the prevalence of Salmonella in captured
and cultured shrimp 14.44% and 11.11%, respectively, and Salmonella Newport was
the highest (47.83%) among the deferent Salmonella serovars followed by Salmonella
Weltevreden 8.7% (Kamalika et al., 2008). Samples of processed and unprocessed
shrimp from processing plant in Nigeria was analyzed and revealed the presence of
Bacillus spp., Salmonella spp., Shigella spp., Enterobacter spp., Micrococus spp., E.
coli, Flavobacterium spp., Staphylococus aureus, Pseudomonas spp., Rhizopus spp.,
Aspergillus flavis, Aspergillus formigatus, Mucor mucido, and Sacchromyces spp., but
no Vibrio spp. were isolated. This study showed a level of contamination by
pathogenic bacteria that is hazardous for the consumer’s health (Okonko et al., 2008).

10
According to Feldhusen (2000), indigenous bacteria found at low levels in
seafood pose an insignificant hazard to the public health if the product is cooked
adequately. But a few bacteria associated with faecal contamination of seafood
(Salmonella, Campylobacter, Listeria, Yersinia, E. coli) continue to cause a large
scale health threat through seafood consumption. This is worse when people have
traditions of eating raw or under cooked seafood like in Japan. Food borne diseases
lead to a significant morbidity and mortalities annually, even in developed countries
where food safety is well regulated. In the United States seafood ranked third on the
list of products that cause food borne illnesses during 1983- 1992 (Lipp and Rose,
1997). Over the past 20 years 4% of shell fish associated outbreaks were from
bacterial pathogens of faecal contamination, while naturally occurring bacteria
accounted for 20% of shellfish-related illnesses and 99% of deaths. Most of the
indigenous bacteria belong to the genera’s of Vibrio, Aeromonas and Plesiomonas
(Lipp and Rose, 1997). The Center for Disease Control and Prevention (CDC)
estimates that approximately 76 million new cases of food-related illnesses and
resulting 5,000 deaths and 325,000 hospitalizations occur in the United States each
year (Group, 2010).
Food borne illnesses has a significant impact on the economics of the public
and private sectors. A calculation of precise figures for the economic impact is
difficult with a high incidence of food borne related illnesses. The evaluation of cost
at a national level in Canada and United States based on the available data showed
that company losses and legal action are much higher than the medical/
hospitalization expenses, lost income or investigational costs. It was estimated that
one million acute bacterial food borne illnesses occur in Canada and 5.5 million cases
in US cost nearly $1.1 billion and $7 billion, respectively every year (Todd, 1989).
FDA estimates the economic impact of food borne illnesses from health related costs
by the sum of medical cost and losses to quality of life (loss of life expectancy, pain
and suffering functional disability). Using CDC data, reports estimate that food borne
illness costs related to produce alone are almost $ 39 billion per year in the US
(Group, 2010). In Australia there has been an increasing incidence of food borne
illnesses caused by E. coli O157:H7, Campylobacter, Salmonella, Hepatitis A,

11
Listeriosis, and other pathogens. Annual estimated costs for these food borne
outbreaks and illnesses is Au. $ 2.46 billion (Khandaker and Alauddin, 2005).
Consumer safety and the economic impact due to food borne pathogens in the
seafood forced the importing countries to impose microbiological criteria on seafood.
Each year the major importing countries of fish and fish products reject or detain
imports due to the presence of microbial pathogens. For the European Union, Vibrio
spp. and Salmonella accounted for 66% of the detention of imports during 1992-2002
and shrimp was dominant among seafood products that cause detention cases. FDA
categorized the presence of Salmonella, Listeria, Shigella, Hepatitis A and general
term bacteria on seafood detention (FAO, 2005). Regulatory requirements for the
absence of Salmonella has been established for cooked/ ready to eat and raw shrimp
in EU, Australia, New Zealand, US and Hong Kong, while ICMSF suggests that
Salmonella should not be detected in 25g raw or cooked shrimp (Wan Norhana et al.,
2010). Rejection of imports causes financial losses to the export countries.
2.5 Prevalence of Vibrio in the shrimp production chain
Vibrio species are indigenous to the marine and estuarine environment
(Bhaskar et al., 1998) and their presence in the shrimp production chain is to be
expected. Several studies done in shrimp producing countries showed the prevalence
of Vibrio in shrimp culture environments and also the retail marketed shrimps (Table
3). Only a few Vibrio species isolated are pathogenic to human and some are
pathogenic to the shrimp itself. Gopal et al. (2005) investigated the prevalence of
Vibrio in on the east and west coast of India and found Vibrio in water, shrimp and
sediment samples. Vibrio alginolyticus (3-19%) V. parahaemolyticus (2-13%) V.
harveyi (1-7%) and V. vulnificus (1-4%) were the predominant species found. The V.
cholera found was negative for the cholera toxin and from V. parahaemolyticus, 2 out
of 47 isolates were tdh positive and one contained the trh gene. Similarly, in a study
done in Karnataka, India, by Bhaskar et al. (1998), all samples of sediment, water,
shrimp, clam meat and formulated feed were contaminated with Vibrio spp. Vibrio
alginolyticus was the most commonly isolated species found in shrimp and sediment.
V. cholerae was found at a very low level but frequently in formulated feed.

12
According to the author it may be due to excessive human handling. In Sri Lanka
Jayasinghe et al. (2008) identified 12 species of Vibrio in shrimp using biochemical
tests from two shrimp farms located in the West and Northwest coastal area.
Aeromonas hydrophelia, V. parahaemolyticus, V. carchariae or V. harveyi and
Plesiomonas shigelloides were the predominant species found among them.
Investigations done in Thailand reported a detection rate of 2% of V. cholerae O1 and
33% of V. cholerae non O1 in coastal water, pond water and shrimp (Dalsgaard et al.,
1995 a).
In addition to shrimp culture environments, there are reports of Vibrio in fresh
and processed shrimp collected from retail markets. Investigations done in Thailand
revealed the presence of Vibrio in samples of raw seafood, processed seafood, ready-
to-eat seafood and shortly boiled seafood available in food markets and supermarkets.
V. alginolyticus was the most frequently found species followed by V.
parahaemolyticus, V. cholera, V. mimicus and V. vulnificus in that order (Chitov et al.,
2009 b). Jonnalagadda and Bhat (2004) found a 5% prevalence of Vibrio spp. in
shrimp purchased from the retail market in India.
Table 3: Prevalence of Vibrio spp. in the shrimp production chain
Point of
sampling
Shrimp culture
environment
Type of
sample
Shrimp
Sediment
Pond water
Origin of
sample
India (East
&West coast)
Shrimp Iran (South
coast)
Coastal
water
Sediment
Pond water
Shrimp
Thailand
(Southern)
No. of
sample
Vibrio spp. found Reference
360 V. parh, V. alg,
V. cho, V. vul,
V. har, (14 Vibrio
spp.)
770 V. parh, V. dam, V.
alg, V. flu
158 V. cho O1
V. cho non O1
Gopal et al.,
2005
Hosseini et al.,
2004
Dalsgaard et al.,
1995 a

Coastal
water
Shrimp
Bangladesh
(Cox bazaar)
Retail Market Shrimp India
(Hyderabad)
Seafoods
(Shrimp)
13
5 V. me, V. alg, V.
ner,
V. har
Sri Lanka 6 V. parh, V. alg,
V. cho, V. vul,
V. har. (12 Vibrio
spp.)
Thailand
(Chiang Mai)
35 Vibrio spp. not
specified.
118 V. alg, V. parh,
V. cho,
V. mi, V. vul
Rahman et al.,
2010
Jayasinghe et
al., 2008
Jonnalagadda
and Bhat, 2004
Chitov et al.,
2009 a
V. alg=V. alginolyticus, V. cho=V. cholerae, V. flu=V. fluvialis, V. fur=V. furnissii, V.
hol=V. hollisae, V. me=V. metschnikovii, V. mi=V. mimicus, V. parh=V.
parahaemolyticus, V. vul=V. vulnificus, A. hy=A. hydrophilia, P. sh=P. shigelloides,
V. har=V. harveyi, V. ner=V. nereis, V. dam=V. damsela
2.6 Importance of shrimp safety in the industry
2.6.1 Public health importance of Vibrio in shrimp
From a public health point of view, concerns about shrimp safety are
increasing with the expansion of the shrimp trade. Most seafood poisoning in the
world is caused by the Vibrio spp. originates with the consumption of fresh and
processed seafood. But the ubiquitous nature of Vibrio makes it impossible to obtain
seafood free from that organism. International trade facilitates the spread of
pathogenic micro-organisms, including Vibrio spp. throughout the world. With the
high demand of shrimp, the industry tends to intensively use antibiotics to control
diseases and to improve production. This leads to develop multi-resistant pathogenic
bacteria (Wan Norhana et al., 2010). A study done in Delhi, India during 2001 to
2006 in hospitalized patients, found V. cholerae E1 Tor Ogawa (54.6%) more
common than serotype Inaba (32.5%). But during 2004 to 2006 V. cholerae Inaba
emerged as the predominant serotype and showed a high resistance to nalidixic acid,

14
furazolidone and cotrimazole and also for cifrofloxacine with MIC>4mg/ml which is
relatively high (Das et al., 2008). Such a serotype change (Ogawa to Inaba) may be
due to pre-existing Ogawa antibodies or perhaps due to antimicrobial selection
pressure. From deferent aquatic locations in Kerala, South India, multi-resistant non
O1 and non O139 V. cholerae strain were isolated and the majority of strains (59%)
were found to be multi-drug resistant (Jagadeeshan et al., 2009). Climatic changes and
global warming impose a negative impact on the water and food borne diseases
caused by micro-organisms. As shrimp is always produced in water and water is used
for processing, these changes affect the shrimp safety. A study done in Bangladesh
showed that the high frequency of cholera outbreaks during the El Nino Southern
Oscillation, which leads to increased surface sea water (Pascual et al., 2000). A
similar study carried out in Africa also confirms the relationship between climatic
change and cholera epidemics (Constantin de Magny et al., 2007). The association
between the El Nino events and records of V. parahaemolyticus infection analyzed
during 1994-2005 time period in Peru identified the El Nino episodes as a reliable
vehicle for the introduction and propagation of Vibrio in South America (Martinez-
Urtaza et al., 2008). With an expanding population of highly susceptible people due to
aging, malnutrition, immune-compromisation and illnesses such as diabetes, the need
for a better understanding of food safety is crucial (Wan Norhana et al., 2010).
Food borne outbreaks cause economic burdens on patients and their families
through treatment costs, loss of productivity, suffering and the risk of death, and also
affect the public health systems of the country financially (Suzita et al., 2009). Food
borne illnesses due to Vibrio are reported throughout the world and are largely caused
by the consumption of seafood. Seven epidemics of cholera have been recorded since
1817 and the 8 th one is in progress (Table 4). During 1992, the epidemic of V.
cholerae O139 originated in the Bengal region of India and spread through India,
eventually to Bangladesh, Nepal, Burma, Pakistan, Thailand, China, Malaysia and
Saudi Arabia. Imported cases were reported in the United Kingdom and the United
States. Some suggest it as 8 th cholera pandemic due to its rapid spread within a 2 year
period (Kaysner, 2000).

Table 4: Cholera pandemics
Pandemic Dates Origin Biotype
I. 1817-1823 Indian subcontinent -
II. 1829-1851 Indian subcontinent -
III. 1852-1859 Indian subcontinent -
IV. 1863-1879 Indian subcontinent -
V. 1881-1896 Indian subcontinent -
VI. 1899-1923/5 Indian subcontinent Classical
VII. 1961-Present Indonesia O1 classical,
15
O1 E1 Tor
VIII. 1992-Present Indian subcontinent O139 serotype
V. parahaemolyticus is the most common cause of food borne disease in the
Asian region. In Japan, it has been implicated as a cause of at least a quarter of the
total food borne diseases (Feldhusen, 2000). In Korea, food borne outbreaks due to
Vibrio from 2003 to 2006 were 80, and 2261 human infections were recorded (Lee et
al., 2008 a). Since 1996 V. parahaemolyticus O3:K6 is considered to be the first
pandemic strain due to its spread from India to many countries within a short period
of time. Western countries also experienced gastroenteritis due to V.
parahaemolyticus (Cabanillas-Beltrán et al., 2006).
2.6.2 Regulatory perspective of Vibrio in shrimp
Japan, the European Union and the United States are the largest seafood
importers and account for 82% of the total volume of seafood imports in the world
(FAO, 2005). Increasing awareness and demand of consumers for safe and high
quality food forced the governments to impose strict regulations and rules on food

16
safety to assure safety of the public health. Importing countries imposed different
quality and safety policies on imported food and it leads to border control, rejection,
or detainment of the product. This leads to direct and indirect financial losses to the
exporters. Seafood becomes the most common product that causes notification on
import alerts. The presence of pathogenic micro-organisms is one of the common
causes for detention (Wan Norhana et al., 2010).
The EU committee on Rapid Alarm System for Food and Feed (RASFF)
showed the highest notification of Vibrio on fish, crustaceans and mollusks during
2001 to 2003 and accounted for 29.3% out of total food groups. Out of seafood types
75% of the cases were due to frozen shrimp. During 1999-2002 border cases reported
in the EU regarding microbial risk, Vibrio spp. showed highest occurrence (39.8%)
followed by the Salmonella (27.7%). The EU countries do not have harmonized
criteria for Vibrio organisms (FAO, 2005). Meanwhile import detention by the Food
and Drug Administration (FDA) in United States for seafood products accounted for
almost 27% of the total detentions in 2001 (Wan Norhana et al., 2010). Investigations
carried out on seafood imported from Hong Kong, Indonesia, Thailand and Vietnam
to Taiwan revealed 45.9% of samples positive for V. parahaemolyticus. The incidence
rate in shrimp was high (75.8%) when compared with other seafood such as crabs,
snails and lobsters (Wong et al., 1999).
Several countries and food authorities set microbiological criteria on raw
seafood and ready-to-eat seafood including shrimp to safeguard their consumers
(Table 5). A regulatory requirement for the absence of V. cholerae has been
established in Hong Kong, the United States, International Commission of
Microbiological Specification for Food (ICMSF), the European Union, the United
Kingdom and Australia. Only the FDA stated that the absence of any serotype of V.
cholerae O1 or non O1 as a criteria to remove the food from the food chain. Presence
of V. parahaemolyticus in the food is accepted in all countries but they set different
accepted levels. The accepted level of V. parahaemolyticus in raw shrimp at time of
sale should be less than 100 cfu/g in Netherland, and raw, frozen and cooked require
similar values (

17
parahaemolyticus should be Kanagawa positive or negative and greater than 10 4 cfu /g
to remove the product from the food chain. Vibrio vulnificus is only focus by the
FDA. Shrimp that have lethal pathogenic organisms are rejected or recalled from the
market.
Table 5: Microbiological criteria for Vibrio spp. in shrimp and shrimp products.
Country/food
authority
Microbiological
criteria/max. limits
Raw shrimp(Fresh/frozen) RTE/cooked shrimp
United States - V. cholerae: presence of
Australia/New
Zealand
V. cholerae/g: n=5, c=0, m=0
toxogenic O1 or non O1
V. parh: levels =

18
Today export countries must adapt those rules and regulations to maintain
trade opportunities. It appears that many countries, especially the poorest may face a
major problem in meeting food safety standards that are obligatory in a number of
importing countries.
2.7 Vibrio
2.7.1 History and Morphology
The first Vibrio spp. identified was Vibrio cholera, discovered in 1854 by the
Italian physician Filppo Pacini during an outbreak and subsequent investigation in
Florence. Pacini detected V. cholerae in all intestinal mucosal samples of fatal
victims. John Snow (1813-1858) studied the epidemiology of cholera in several cities
of England and found that cholera is spread by the contaminated water and suggested
pure water for drinking (Thompson et al., 2004).
According to the Bergey’s Manual (Holt et al., 1994) this bacteria is in the
family Vibrionaceae and genus Vibrio and has characteristics of straight or curved
rods, (0.5-0.8µm in width and 1.4-2.6 µm in length), motility by one or more polar
flagella, Gram negative, chemoorganotropic, facultative anaerobic, mesophilic,
oxidase positive and sensitive to the vibriostatic agent O/129 (except a few species).
Most species grow well at 37°C. Vibrio is distributed worldwide and is found in sea
water, fresh water, brackish water and associated with aquatic animals, sediments and
feeds (Bhaskar et al., 1998). Large numbers of Vibrio and Photobacterium attach to
the external surface of the zooplankton and make bio films. Because of this close
association with zooplankton, it is assumed that cholera outbreaks are linked to
planktonic blooms and rising sea water temperature due to global warming
(Thompson et al., 2004).

19
2.7.2 Biochemical characteristics of Vibrio
Biochemical characteristics of Vibrio are used to differentiate the food
associated with Vibrio pathogens including Aeromonas hydrophila and Plesiomonas
shigelloides that belong to the family Vibrionaceae (Table 6) (Kaysner, 2000).
Thiosulfate citrate-bile salt agar (TCBS) is a selective medium for the isolation of
Vibrio (Thompson et al., 2004). Vibrio organism’s ability to ferment sucrose and a
colour change of the colony helps to differentiate Vibrio spp. Sucrose positive, yellow
colonies on TCBS agar are characteristics of V. cholerae, V. fluvialis, V. furnissii, V.
alginolyticus, V. metschnikovii. While V. parahaemolyticus, V. mimicus, V. vulnificus
and V. hollisae are sucrose negative and produced green colour colonies.
Table 6: Biochemical characteristics of the Vibrionaceae commonly encountered
in seafood
V.
alg
V. cho V.
flu
V.
fur
V. hol V. me V. mi V.
parh
V. vul A.
TCBS agar Y Y Y Y NG Y G G G Y G
Oxidase + + + + + - + + + + +
Arginine
dihydrolase
Lysine
decaboxylase
Growth in
(w/v):
- - + + - + - - - + +
+ + - - - + + + + V +
0% NaCl - + - - - - + - - + +
3% NaCl + + + + + + + + + + +
6% NaCl + - + + + + - + + + -
8% NaCl + - V + - V - + - - -
10% NaCl + - - - - - - - - - -
Growth
at 42 o C
Acid from:
+ + V - nd V + + + V +
Sucrose + + + + - + - - - V -
D-Cellobiose - - + - - - - V + + -
Lactose - - - - - - - - + V -
Arabinose - - + + + - - + - V -
D-Mannose + + + + + + + + + V -
hy
P.
sh

20
D-Mannitol + + + + - + + + V + -
ONPG - + + + - + + - + + -
Voges-
Proskauer
+ V - - - + - - - + -
Y= yellow, G= green, NG= no or poor growth, nd= not done, V=variable among
strains, R=resistant, S=susceptible
V. alg=V. alginolyticus, V. cho=V. cholerae, V. flu=V. fluvialis, V. fur=V. furnissii, V.
hol=V. hollisae, V. me=V. metschnikovii, V. mi=V. mimicus, V. parh=V.
parahaemolyticus, V. vul=V. vulnificus, A. hy=A. hydrophilia, P. sh=P. shigelloides
Currently, 72 species are included in the Vibrio genus and 12 species are
commonly isolated in human patients. From these 12 species, V. cholera, V.
parahaemolyticus and V. vulnificus are the most harmful to humans. Other Vibrio spp.
isolated from humans are V. alginolyticus, V. fluvialis, V. mimicus, V. (Grimontia)
holisae, V. (Photobacterium) damsel, V. furnssii, V. cincinnatiensis, V. harveyi, and
V. metschnikovii.
2.7.3 Growth parameters of Vibrio spp.
Table 7 shows the growth limiting factors of major human pathogenic Vibrio spp.
Table 7: Growth limiting factors of Vibrio (FAO, 2003)
Species Temperature
o
C
pH aW NaCl (%)
Min Max Min Min Max
V. cholerae 10 37 5.0 0.97 8
V. parh 5 37 4.8 0.93 8-10
V. vulnificus 8 37 5.0 0.96 5
V. parh = V. parahaemolyticus

21
2.7.4 Genotypic characteristics of Vibrio
Researchers have investigated the phenotypic and genotypic characterization
of the Vibrio organism using new molecular technology based on the ribotyping and
polymerase chain reaction technique such as amplified fragmented length
polymorphism (AFLP), fluorescence in situ hybridization (FISH) or multi locus
sequence typing (MLST) (Thompson et al., 2004). Identification of toxigenic genes
and serogroups (Dalsgaard et al., 2001, Janssen et al., 1996, Raghunath et al., 2008),
genetic diversity (Jiang et al., 2000, Sawabe et al., 2002, Zo et al., 2002), ecological
interaction and relatedness of clinical and environmental isolates (Zo et al., 2002) of
Vibrio has been carried out with the help of new techniques. Toxigenic Vibrio
cholerae O1 samples from the aquatic environment and human intestines isolated
from Bangladesh were analyzed by enterobacterial repetitive intergenic consensus
sequence-PCR, optimized for profiling by using the fully sequenced V. cholerae E1
Tor N16961 genome. A study found a similar composition of environmental toxigenic
V. cholerae and the V. cholerae that causes the endemic cholera. Authors conclude
that the spatial and temporal fluctuation (seasonal fluctuations in the environment,
introduction of new strain) in the composition of the toxigenic V. cholerae population
in the aquatic environment can cause shifts in the dynamics of this disease (Zo et al.,
2002). An investigation carried out by Jiang et al. (2000) detected V. cholerae isolates
in Chesapeake Bay coastal waters. The genetic diversity analyzed by applying AFLP
fingerprinting revealed the link between the shift of the genotype and environmental
changes such as water temperature.
MLST was developed recently and considered as most reliable molecular tool
for the epidemiology. This method is based on the sequence analysis of housekeeping
(HK) genes of the organism. Higher discriminatory power, accuracy and portability of
the data, ease of performance, and reproducibility are major advantages of this
method (Thompson et al., 2004). Nucleic acid sequences are stored in a public data
base that can be easily accessed via internet (http://www.mlst.net or
http://pubmlst.org) (González-Escalona et al., 2008). Scientist applied this method to
determine the global epidemiology of bacterial pathogens. González-Escalona et al.

22
(2008) analyzed the genetic relatedness and geographical distribution of V.
parahaemolyticus from the environment and clinical strains using seven house
keeping genes (dnE, gyrB, recA, dtdA, pntA, pyrC, tnaA).
2.7.5 Molecular and genetic characteristics of environmental Vibrio
Vibrio species are widely distributed in the aquatic environment and are
considered as autochthonous bacteria in the estuarine and marine waters (Nishibuchi
and Kaper, 1995). Scientists isolated and identified this organism in the marine water
(Faruque et al., 2004, Nair et al., 1988), shrimp and oysters (Dalsgaard et al., 1995 a,
Gopal et al., 2005, Sobrinho et al., 2010), sediments (Bhaskar and Setty, 1994),
plankton (Lipp and Rose, 1997), and also from the seafood (Chitov et al., 2009 b,
Sujeewa et al., 2009). Many investigations were carried out to identify genetic and
phenotypic differences between clinical and environmental vibrios.
V. cholerae serogroups O1 and O139 caused the epidemics of cholera. But
other non O1 and non O139 serogroups can cause sporadic diarrhoea. Environmental
strains mainly belong to V. cholerae serogroup non O1 and non O 139 (Faruque et al.,
2004). Clinical strains carry the virulence factors for cholera toxin (CT) and toxin-
coregulated pilus (TCP) which is central to the disease process and provide adhesion
for the toxins respectively (Chakraborty et al., 2000). Studies found that
environmental strains of V. cholerae rarely carried CT and TCP. Water samples from
two major rivers of Bangladesh were investigated for the presence of V. cholerae and
their virulence associated genes. V. cholerae non O1 and non O139 was found in
89.9% (116/125) and 3.2% (4/125) were V. cholerae O1 (Faruque et al., 2004). These
authors found one V. cholerae O1 carrying the TCP and CT gene (0.8%) in those
environmental samples. Nair et al. (1988) carried out an investigation on V. cholerae
non O1 from environmental sources in India, to describe toxin profile. They examined
for the production of CT, shiga-like toxin (vero toxin), heat stable enterotoxin and
haemolysins. Two of the strains (0.5%) produced CT; none of them produced shiga-
like toxin or heat stable enterotoxins. Haemolytic activity was observed in 89.7% of
the strains. The authors concluded that only a small percentage of environmental V.
cholerae non O1 has the ability of causing cholera like symptoms (Nair et al., 1988).

23
Clinical strains of V. parahaemolyticus have the ability to produce
thermostable direct haemolysin (TDH) and TDH related haemolysin (TRH) and can
cause gastroenteritis in humans (Su and Liu, 2007, Vongxay et al., 2006). Most of the
tdh positive strains of V. parahaemolyticus exhibit the Kanagawa phenomenon, an
enzymatic lysis of red blood cells on Wagatsuma blood agar plates (Su and Liu, 2007,
Vongxay et al., 2008). Several investigations found that only 1-3% of the
environmental strains of V. parahaemolyticus exhibit the toxic activity (Lee et al.,
2008a, Vongxay et al., 2006, Sobrinho et al., 2010), (Table 8). But recent reports on
the presence of virulence genes in environmental strains of V. parahaemolyticus
found an increasing percentage of tdh and trh gene-carrying strains (Deepanjali et al.,
2005, Raghunath et al., 2008, Sujeewa et al., 2009, Zimmerman et al., 2007) (Table
8). Those studies showed that, trh-bearing V. parahaemolyticus are more frequently
distributed in tropical seafood than tdh-bearing V. parahaemolyticus.
V. vulnificus is an opportunistic human pathogen (Cañigral et al., 2009) and
unlike V. parahaemolyticus and V. cholerae, environmental and clinical strains of V.
vulnificus showed no difference in the potential to produce toxins. Human V.
vulnificus infections shows clinical signs of wound infection, fever, chills,
hypotension, bulbous like skin lesions which are rapidly progressive and 50%
mortality in septicaemia conditions. The polysaccharide capsule and the production of
siderphores by this organism are considered as virulence factors (Starks et al., 2000).
Tison and Kell (1986) studied the virulence of V. vulnificus isolated from the marine
environment. These authors found no difference in their production of virulence
factors, both in vitro (cytolysin and cytotoxin) and in vivo (mouse pathogenicity)
among clinical and environmental isolates of V. vulnificus. Similar to this study,
Wong et al. (2005) also found that environmental strains of V. vulnificus exhibit a
similar virulence to clinical strains in mice. These data were supported by Stelma et
al. (1992).

24
Table 8: Prevalence of tdh and trh genes of V. parahaemolyticus in
environmental samples.
Country/
place
Type of
samples (no
of samples)
Prevalence
% of V.
parh.
Presence of
tdh gene in
%
Presence of
trh gene in
%
References
Korea Raw oysters - 0 1.38 Lee et al., 2008
(72)
a
Brazil Raw oysters 99.2 0.8 0 Sobrinho et al.,
(123)
2010
India Shrimp, 2-13 4 2.12 Gopal et al.,
sediment,
water
2005
China Samples from 14.3 5.8 0 Vongxay et al.,
seafood
processing
line (258)
2006
Malaysia Frozen
51 15 7 Sujeewa et al.,
shrimp (60),
2009
live
(50),
shrimp
sediment
(67),
(74).
water
Southwest Raw Oysters 93.9 10.2 59.3 Deepanjali et
coast of India
al., 2005
Southwest
coast of India
Northern
Gulf of
Mexico
Oysters (44),
clams (14),
fish (9),
shrimp (16),
seafood (83)
Oysters (32)
Water (102)
V. parh. =V. parahaemolyticus
89.2 8.4 25.3 Raghunath et
al., 2008
56/44
78/30
56
44
78
30
Zimmerman et
al., 2007

2.8 Human pathogenic Vibrio
2.8.1 Vibrio cholerae
25
Cholera has been recognized as a fatal disease since 1817 and six pandemics
have already swept over the world (Suzita et al., 2009) and the 7 th and 8 th pandemics
are still progressing (Kaysner, 2000). Vibrio cholera is transmitted to humans via
contaminated food, water, raw seafood or direct infection from food handlers (Suzita
et al., 2009, Thompson et al., 2004). Cholera is characterized by profuse acute
diarrhoea (rice water), vomiting, dehydration and death within 24 hours if left
untreated (Suzita et al., 2009). The pathogenesis of this organism in the intestine is
characterized by adherence to the epithelium and production of an enterotoxin
(cholera toxin) which leads to intense watery diarrhoea. In addition to the toxin, the
toxin coregulated pilus is essential for the micro colonization of the intestinal
epithelium (Thompson et al., 2004).
Currently more than 200 serogroups of V. cholera are identified based on the
somatic O antigen. Vibrio cholera O1, O139, non-O1, non-O139 and O141 are the
deferent O strains that cause diarrhoea throughout the world. V. cholerae O1 has two
serogroups Inaba and Ogawa and two biotypes namely, Classical and El Tor (Ganesh
et al., 2010, Suzita et al., 2009). Serogroup O1 and O139 are responsible for the
epidemics and pandemic occurrences of cholera and non-O1 and non-O139 are less
virulent forms found in patients. V. cholerae O141 causes cholera-like diarrhoea and
bacteraemia in the United States (Crump et al., 2003).
2.8.2 Vibrio parahaemolyticus
Vibrio parahaemolyticus is a Gram negative, mesophilic and halophilic rod-
shaped bacterium found in the estuarine environment, especially in shellfish, coastal
fish and seafood. Consumption of raw, under cooked or contaminated shellfish, fish
and seafood is the method of transmission of this bacterium to humans. It causes
gastroenteritis, nausea, watery diarrhoea, vomiting, abdominal cramps, low grade
fever, chills and sometimes bloody diarrhoea (Cabanillas-Beltrán et al., 2006). V.
parahaemolyticus produces toxins: thermo-stable direct haemolysin (TDH), encoded

26
by the tdh gene and the TDH related haemolysin (TRH) encoded by the trh-gene. The
production of the TDH toxin can be detected biochemically by the Kanagawa
reaction, involving beta-haemolysis on Wagatsuma blood agar. The trh-gene can be
detected by PCR (Thompson et al., 2004).
The first pandemic strain of V. parahaemolyticus O3:K6 appeared in Calcutta,
India and rapidly spread to many countries since 1996. During 2003-2004 the same
serotype O3:K6 appeared in Mexico and caused gastroenteritis affecting more than
1230 human cases (Cabanillas-Beltrán et al., 2006).
2.8.3 Vibrio vulnificus
Vibrio vulnificus is a halophilic bacteria naturally found in the estuarine and
coastal waters. Gastroenteritis, severe necrotization of the soft tissue and primary
septicaemia with a high mortality rate are the clinical signs. Consumption of V.
vulnificus contaminated food and exposure to the contaminated water causes the
illness (Cañigral et al., 2009). Septicaemia occurs mainly in immunosuppressed
people and in patients with high levels of serum iron due to liver disease or genetic
disorders. The primary virulence factor of V. vulnificus, the capsular polysaccharide,
plays an inflammatory role within the human body (Thompson et al., 2004). V.
vulnificus biotype 1 can be lethal and biotype 2 can be an opportunistic pathogen to
humans. Biotype 2 is pathogenic in Asian and European eels. The prevalence of this
bacterium is strongly correlated with the water temperature (Feldhusen, 2000).
2.9 Detection of virulence genes of Vibrio
V. cholerae serogroup non O1 and non O139 strains have not been associated
with epidemics but they can cause sporadic diarrhoea (Chakraborty et al., 2000).
Clinical strains of V. cholerae (O1 and O139) exclusively carrying the virulence
factors of cholera toxin (CT) and toxin-coregulated pilus (TCP). These factors are
encoded by the ctx and tcpA genes (Chakraborty et al., 2000, Srisuk et al., 2010).
Identification and detection of V. cholerae and its virulence is often achieved by
traditional biochemical methods which was labour and time consuming. But some

27
Vibrio spp. display similar biochemical characteristics and it limits the proper
identification and characterization of Vibrio spp. (Tamrakar et al., 2006). Scientists
have developed PCR based techniques for the detection of toxigenic strains based on
the cholera toxin (ctx) gene, and the toxin coregulated (tcpA) gene (Keasler and Hall,
1993, Rivera et al., 2003).
V. parahaemolyticus has been recognized as major pathogenic agent of
gastroenteritis associated with the consumption of seafood (Nishibuchi and Kaper,
1995). But not all V. parahaemolyticus strains are carrying the virulence capacity
(Raghunath et al., 2008). Clinical strains are categorized into two groups according to
their ability of haemolysing Wagatsuma blood agar. This is named as Kanagawa
phenomenon (KP). Most of the clinical strains of V. parahaemolyticus are haemolytic
(KP + ). This haemolysin was identified as thermo stable direct haemolysin (TDH),
because it was not inactivated by heating at 100°C for 10 min. Kanagawa
phenomenon negative strains were isolated and named as TDH-related haemolysin
(TRH) and its activity is lost when heated at 60°C or higher temperature for 10 min.
(Honda and Iida, 1993). Clinical strains of V. parahaemolyticus most often produce
TDH or TRH haemolysin and carry the gene for tdh and trh respectively (Nishibuchi
and Kaper, 1995, Raghunath et al., 2008). Both TDH and TRH strains have similar
biological, immunological and physiochemical characteristics and they are composed
of 165 amino acids and their amino acid sequence homology is about 67% (Honda
and Iida, 1993). These data suggested that trh and tdh genes probably evolved from a
common ancestor. Researchers identified the tdh gene in some strains of V. mimicus,
V. cholerae non-O1 strains from the marine environment and occasionally from
diarrhoea samples while all the strains of V. hollisae were positive for tdh gene
(Honda and Iida, 1993, Nishibuchi and Kaper, 1995).
Identification of virulent strains of V. parahaemolyticus has been done with
the conventional methods, such as detection of haemolysin in Wagatsuma agar
containing washed human or rabbit erythrocytes, immunological methods (reverse
passive haemagglutination, ELISA) (Honda and Iida, 1993). But for the detection of
TRH there are no available commercial methods. Therefore identification of virulence

28
genes by DNA based molecular techniques such as PCR and colony hybridization
methods are more important. Researchers found the PCR method as a more reliable,
rapid and sensitive method compare to conventional microbiological assays (Bej et
al., 1999, Raghunath et al., 2008).
2.10 Prevention and control of Vibrio in shrimp production chain
2.10.1 Pre harvest stage
2.10.1.1 Biosecurity measures
With an expanding aquaculture industry, appropriate techniques are required to
mitigate diseases. With new technologies, biosecurity measures can easily be adapted
for small or large scales farms. Biosecurity is defined as the practices that reduce the
probability of pathogen introduction and control the subsequent spread from one place
to another. The primary goal of biosecurity in shrimp farming is to prevent entry of
infectious organisms into the farm. Possible carriers are, (i) infected host (post larvae,
brood stock); (ii) non-host biological carriers (contaminated water); (iii) inanimate
objects contaminated with pathogens (vehicles, human, nets) (Lotz, 1997). A sound
biosecurity program of a shrimp farm includes disease prevention, disease monitoring,
management of disease outbreak, cleaning, disinfection and general security
preventions.
Perera et al. (2008) recommended measures can be taken to minimize the
input of pathogens into the farm. For example, stocks (brooding/post larvae) can be
obtained from populations of specific pathogen free or specific pathogen resistance
stock or populations subject to monitoring and known to be in good health, or
quarantined and tested or treated to reduce the risk. Pathogen transmission by feed can
be controlled by providing commercial pelletized feed that are subjected to a degree
of pathogen inactivation by heat generated through the process of extrusion and pellet
drying (Perera et al., 2008). Water is the major non-living conveyer of the pathogens.
Pathogens may be present in the incoming water because of the presence of natural

29
host and effluent from the contaminated farms (Lotz, 1997). Intake water can be
purified by screening and filtering to reduce the formites and carrier organisms. Risk
associated with water can be reduced by application of UV, ozone or chemicals before
use (Perera et al., 2008). A study found that application of alternating low-amperage
electric treatments to effluent sea water to inactivate the V. parahaemolyticus while
generating only low levels of chlorine (Park et al., 2004). This method was able to
overcome the problem of chlorine generation that usually results from treatment with
continuous direct current. Farm equipment, such as aeration, harvest nets, containers,
foot wear and vehicles need to be disinfected using suitable disinfectants such as
sodium hypochlorite, iodophors and sodium chloride. Periodically testing for the
specific diseases can carried out for the early detection and quick response and
prevention of disease outbreaks. Training of farm staff is essential for awareness of
the potential risks and biological principles in biosecurity measures (Perera et al.,
2008).
In Sri Lanka, biosecurity measures are monitored by the National Aquaculture
Development Authority (NAQDA). Screening of brood stock for white spot disease is
carried out before being issued to the farmers. If the farm is infected with a disease,
officers inform farmers to harvest non-infected farms using nets, after that affected
farms disinfected using pesticides to destroy the infected shrimps. Water is released
after 4-7 days in to the sediment areas (Weerakoon, 2007). Sometimes the agency
delays the restocking of the post larvae for the next crop in affected zone. Even
though farmers gain knowledge on biosecurity, economic issues restrict the farmer’s
ability or willingness to invest in biosecurity (Munasinghe et al., 2010).
2.10.1.2 Application of probiotics
The use of probiotics is widely prevalent in the aquaculture, especially in
shrimp culture as a means of controlling diseases and improving water quality without
the use of antibiotics and disinfectants (Ma et al., 2009). A probiotic is defined as a
live microbial agent that prevents pathogens from proliferating in the intestinal tract,
on the superficial structures and in the cultural environment of species and that aid

30
digestion, improves water quality, or stimulate the immune system of the host
(Verschuere et al., 2000). Microorganism belonging to photosynthetic bacteria,
Nitromonas spp., Lactobacillus spp., yeast, Bacillus spp., Pseudomonas fluorescens,
Streptococcus faecium, Tetraselmis succia and other bacteria have been tested as
probiotics (Wang et al., 2005).
Several studies have been carried out to determine the effectiveness of
probiotics on aquaculture. Rengpipat et al. (1998) reported the use of Bacillus strain
S11 as a probiotic in black tiger (P. monodon) shrimp culture. After a 100 day feeding
trial with probiotic supplements and non-supplement feed, P. monodon challenged
with pathogenic V. harveyi strain D331 by immersing the shrimp. Ten days later, all
the shrimp groups treated with probiotics showed 100% survival and the control
group had 26% survival (Rengpipat et al., 1998). Similar research was carried out in
shrimp ponds in the Philippines where the losses due to luminas Vibrio (V. harveyi)
are catastrophic. Bacillus spp. used as probiotics and experienced a higher survival
rate (80-100%) in treated farms (Moriarty, 1999). Investigations carried out to
determine the effectiveness of probiotics on white shrimp (P. vannamei) found a
noticeable influence on the shrimp production and an increase in the water quality by
reducing concentration of nitrogen and phosphorous. A study found that the average
count of Bacillus spp., amonifying bacteria and protein mineralizing bacteria were
significantly higher in probiotic treated ponds compared to control ponds.
Furthermore, it increased dissolved oxygen and reduced dissolved reactive-
phosphorus, total inorganic nitrogen and chemical oxygen demand. Shrimp
production and survival rate also increased in treated ponds. The results were average
8215± 265 kg shrimp/ ha and a survival rate of 81.0 ± 6.75% in treated ponds
compared with 4985± 503 kg shrimp/ ha and 48.67± 3.51% respectively in control
ponds (Wang et al., 2005). Ma et al. (2009) tested strains of Lactobacillus spp. JK-8
and JK-11 for their ability to remove pathogenic bacteria and nitrogen compounds.
Cell-free supernatants concentrated from pH-non adjusted JK-8cultures and either
pH-adjusted or non adjusted JK-11 cultures demonstrated remarkable antimicrobial
activities against pathogenic bacteria. Vibrio parahaemolyticus and V. harveyi, were
significantly susceptible and destroy within 30 minute exposure. Complete

31
elimination of Edwardsiella tarda, S. aureus, Salmonella and Shigella were achieved
within 1.5 hrs and E. coli and S. pyrogenes were completely killed within 2-3 hrs
under same condition. Simultaneously, both JK-8 and JK-11 culture facilitated the
removal of different nitrogen compounds (e.g. NH + 4, NO - 2, and NO - 3) up to 400 µM.
2.10.1.3 HACCP application
The use of hazard analysis critical control point (HACCP) as a means of
controlling food borne pathogens in the food industry is gaining acceptance
internationally both by industry and regulatory agencies. Food manufacturers can
identify steps for preventing, controlling or eliminating hazards associated with their
product and minimize the potential food safety problems (Buchanan, 1995). HACCP
in shrimp farms can include all steps from the “farm to table” such as, production and
handling of raw materials, processing operation, processing environment, handling
and storage practices, and distribution activities (Tookwinas and Keerativiriyaporn,
2004). A good aquaculture practice is a prerequisite for the implementing HACCP.
Good aquaculture practices are defined as those practices of the aquaculture sector
that are necessary to produce quality food products, conforming to food law and
regulations. Conducting hazard analysis, determine critical control points (CCP),
establish critical limits, establish a system to monitor control of CCP, establish the
corrective action, establish procedures for verification and establish documentation
are the main 7 steps included in the HACCP system (Reilly and Käferstein, 1997).
Reilly and Käferstein (1997) identified four critical control points in the aquaculture
farming. Those are site selection, water supply, feed supply and production/grow out
stage, and explained the control and verification methods for each step. Farm
management, feed production and farming practices are the CCP that have been
studied by Tookwinas and Keerativiriyaporn (2004) and preventive measures applied
in shrimp farming in Thailand are i). Farm registration; ii). Control the uses of
feed/antibiotics; iii). Monitor residues in products from farm; iv). Mobile unit control
of diseases; use of antibiotics and feed; v). Monitoring the quality of water (both inlet
and outlet); vi). Inspect farm hygiene and post harvest handling practices; vii). Train

32
farmers on good aquaculture practices, safe use of chemotherapeutic agents and good
handling practices.
Verification of HACCP plans for aquaculture production should be carried
out by qualified personnel to verify that the CCPs are satisfactory. Record keeping is
central to implementation in the HACCP system. The preparation of the HACCP plan,
updates, its implementation must be fully documented and records should be kept for
a period of two years and be available for inspection by a regular authority (Reilly and
Käferstein, 1997).
2.10.2 Post harvest stage
2.10.2.1 Post harvest handling
As shown in Table 7, optimum growth temperatures of the Vibrio are 37 °C.
Most of the shrimp producing countries are experiencing high environmental
temperatures that provide suitable temperatures for the growth of Vibrio spp. A study
reported that after harvest; V. parahaemolyticus multiply rapidly at 26 °C showing a
50-fold increase in 10h and 790-fold increase in 26 °C at 24 hours (Gooch et al.,
2002). An investigation was carried out of the post harvest surveillance for V.
vulnificus in post harvest oysters. It revealed the seasonal relationship with the density
of the organism. Low densities were observed in June, increasing through August and
becoming rare by September (La Valley et al., 2008). Therefore, it is most important
to immediately reduce and maintain the temperature of fresh post harvest shrimp at
low levels to prevent the multiplication of Vibrio to infectious levels.
2.10.2.2 Post harvest processing treatments
2.10.2.2.1 Thermal process
Thermal processes such as cold storage, freezing and low temperature
pasteurization have been reported effective in control the multiplication of Vibrio
organism.

A. Cooling and freezing
33
Rapid and efficient cooling to 5 °C will prevent the growth of V.
parahaemolyticus. Vibrio vulnificus is more sensitive to cold storage than V.
parahaemolyticus and reduces with approximately 0.04 log units/day under normal
cold temperature storage. Even though V. cholerae be reduced in cold storage, they
should not be relied on as preventive measures (FAO, 2003). It has been shown that
V. cholerae can survive under chilled and frozen temperatures on shellfish due to
presence or absence of the chitin-containing carapace of shrimp, which has a
cryoprotective effect on the pathogen (Shimodori et al., 1989). Sodium-
metabisulphate is traditionally used in shrimp to control non-microbiological spoilage
known as black spot. A study reported significant decreases (up to 1.8 log units) of V.
cholerae on chilled sodium-metabisulphate- treated, compared to untreated chilled
prawns (Januário and Dykes, 2005).
B. Heating
Vibrio is very sensitive to the heat (Table 8). Boiling temperature is very
harmful to the V. cholerae O in shrimp samples with or without carapace and
complete destruction occurs within 1-2 minutes after exposure to the temperature
(Nascumento et al., 1998). V. parahaemolyticus shows a >7 log reduction in viable
cell count when heated at 55 °C for 2 minutes (Yeung and Boor, 2004). Therefore
low heat pasteurization (50 °C for up to 15 min) can be applied for reduction of
Vibrio in the seafood (Andrews et al., 2003).
Table 9: Heat resistance of Vibrio spp. (FAO, 2003)
Vibrio species Heat resistance (D value)
V. cholerae D55 =0.24 min
V. parahaemolyticus D60 =0.71 min
V. vulnificus D50 =1.15 min (buffer); 0.66 min
(oysters)

2.10.2.2.2 High pressure processing
34
High hydrostatic pressure is a non thermal process that can apply to destroy
pathogenic microorganisms in food and extend shelf life without changing the
appearance and original nutrients (Su and Liu, 2007). Studies have revealed the effect
of high pressure on Vibrio spp. in oysters. Authors reported that to achieve a 5-log
reduction of V. parahaemolyticus in live oysters, the pressure needed to be >350 MPa
for 2 min at temperature between 1 and 35 °C and ≥ 300 MPa for 2 min at 40 °C
(Kural et al., 2008). Vibrio cholerae, V. parahaemolyticus, V. vulnificus in oysters
was tested for high pressure to gain >5-log reduction and observed variability in
sensitivity among species. Vibrio vulnificus was most sensitive to the treatment at 200
MPa (D=26s) and V. cholerae was the most resistant to treatment at 200 MPa
(D=149s). To achieve a >5- log reduction, V. vulnificus required a pressure of 250
MPa for 120s and for V. parahaemolyticus, including V. parahaemolyticus O3:K6
strain required 300 MPa for 180s (Cook, 2003).
2.10.2.2.3 Irradiation
Irradiation is another non-thermal processing method and a low dose of
ionizing radiation has proven to be effective in reducing pathogenic bacteria in food.
A study found that a 1.0 kGy dose was sufficient to produce a 6-log reduction in V.
parahaemolyticus and a dose of 3.0 kGy is effective in inactivating V.
parahaemolyticus in oysters without changing sensory quality and without killing the
oyster (Jakabi et al., 2003). Another study revealed that a Cobolt-60 gamma radiation
treatment at 1-0.75 kGy reduces the V. vulnificus from 10 6 cfu/g to a non detectable
level

2.10.3 Consumer education
35
Consumer behavior is closely related with food borne out breaks. As with
other food borne diseases, Vibrio infection also can be reduced through application of
hygienic food handling techniques. Such practices include, storing seafood at low
temperature, avoiding cross-contamination of ready-to-eat food with uncooked
seafood and thorough cooking to destroy the pathogenic organism (Yeung and Boor,
2004). Individuals with liver or blood disorders and other medical conditions (such as
patients with high level of serum) need to be educated to avoid consuming raw or
undercooked seafood for the prevention of V. vulnificus infection (Kaysner, 2000).