iv

2.1. Vibrio anguillarum

Vibrio anguillarum is a member of genus Vibrio, which consist of 96 species

(http://www bacterio.cict.fr/, updated 2011). Vibrios are highly abundant in aquatic

environments, including estuaries, marine coastal waters, sediments, and aquaculture

ecosystem (Thompson et al., 2004). Being a Gram negative, motile, comma like rods, able to

grow on marine agar and thiosulfate-citrate-bile salt-sucrose agar (TCBS), and chemo-

organotrophic facultative fermentative metabolism nature, nominate the V. anguillarum to be

ranked in the genus of Vibrio. The species name has coined based on the eel fish (Anguilla),

from where this bacteria was first isolated (Austin and Austin, 1999). V. anguillarum was the

first Vibrio known to be fish pathogen, and for many years all the pathogenic vibrios were

ascribed to this taxon, but with the increase interest in aquaculture activities, it became clear

that other distinct species of vibrios were playing role in fish infection such as V. alginolyticus

and V. vulnificus (Colorni, et al., 1981).

2.1.1 Historical perspective

As in the case of cholera in the humans, fish vibriosis was known since centuries as

“red pest”. Red pest caused severe mortality in eels across the Italian coast, during the 18th and

19th centuries. The era of Vibrio isolation started with the isolation of V. cholerae in 1883 by

Robert Koch, from human infection, while aquatic Vibrio isolates such as V. fischeri and V.

splendidus started to be cultured in the late 1880s by Martinus Beijerinck. V. anguillarum

isolated for the first time by Canestrini in 1893, and was designated as Bacterium anguillarum

but later renamed as V. anguillarum in 1909 by Bergman (Elis, 1988; Thompson et al.,

2004).

2.1.2 Taxonomy and nomenclature

v

V. anguillarum is taxonomically classified as bacteria, phylum proteobacteria, class

gamma proteobacteria and family vibrionaceae (Bergey and Holt, 1994). Across the history

there was a lot of debates in regard to the genus to which V. anguillarum classified in. Vibrio

piscum in 1927 and Actinobacter ichthyodermis in 1944 were regrouped under the name

proposed by Bergman in 1909, Vibrio anguillarum (Hendrie et al., 1971). MaCdonell and

Colwell has reclassified this bacteria under new genus Listonella in 1985, based on study the

alignment between the 5S RNA sequences. The new name, Listonella anguillarum was not

widely used in microbial society. Though the International journal of systematic bacteriology

has announced the validity of the new name Listonella anguillarum in list no.20, 1986 but the

recent edition of Bergey’s manual has chosen the name Vibrio anguillarum, while Listonella

was used in parentheses as an option. That was to avoid confusion since genus Vibrio still

required more details to determine its evolutionary heterogeneity (Bergey and Holt 1994).

Austin et al., 1995 and Austin et al., 1997 refused the new classification considering Vibrio as

more suitable choice than Listonella. A scan of the recent papers abundantly including

Colwell, 2005 shows that the name Vibrio anguillarum is highly favorable name than the

Listonella anguillarum.

2.1.3. Typing of V. anguillarum

The first type differentiation of V. anguillarum was developed in 1935 by Nybelin. The

biotype 1 (anguillicida) and biotype 2 (typica) were identified based on the ability of biotype 2

strains to utilize sucrose and mannitol and ability to produce indole; biotype 1 were negative

for these reactions. Biotype 3 was identified latter to be intermediate between the first and

second biotypes. Biotype 3 differed from biotype 2 in its ability to produce indole (Smith,

1961). Development in DNA typing methods have found differences between biotype 1 and

biotype 2 which ultimately reclassified the biotype 2 into new specie V. ordalii (Schiewe and

Crosa, 1981). On the basis of the cross-adsorption reactions of the thermostable somatic

vi antigens, the V. anguillarum is placed into three serotypes (Patch and Kiehn, 1969). In more

exclusive study which included 585 V. anguillarum isolates, serological detection of O antigen

has subdivided V. anguillarum into 10 serotypes (O1 to O10). Interestingly, it also revealed a

correlation between pathogenicity and serological characters, since 71% of the isolates from

cultured fish were belong to serotype O1, and 78% of the isolates from feral fish were

belonging to serotype O2 (Sorenson and Larsen , 1986). An additional 6 serotypes have been

reported by Grisez and Ollevier, 1995 based on lipopolysaccharide antigens.

2.1.4. Distribution

Among all bacterial fish infections, vibriosis is considered the most frequent disease as

seen from figure1 (Tornazo 2004). V. anguillarum have been reported as the causative agents

of fish vibriosis in feral and farmed fish. Over 45 species of fish have been reported to be

infected with V. anguillarum, including the important economical species cultured in

aquaculture such as Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), sea

bream (Sparus aurata), sea bass (Dicentrarchus labrax), ayu (Plecoglossus altivelis), Atlantic

cod (Gadus morhua), saithe (Pollachius virens) and turbot (Scophthalmus maximus) (Larsen

etal., 1994; Bullock, 1987). Shellfish such as Penaeus monodon are also susceptible, however

with a lower incidence (Vaseeharan, 2008). V. anguillarum are also known to affects bivalves

(Bolinches et al., 1986).

vii

Figure 1. Summary of bacterial disease in fish (Tornazo, 2004)

Geographically, V. anguillarum is reported to be globally distributed. It was first

isolated from Baltic Bay (Elis, 1988). Many countries around Mediterranean Sea established a

survey of vibriosis such as, Croatia, Cyprus, Egypt, France, Greece, Israel, Italy, Malta,

Morocco, Portugal, Spain, Tunisia and Turkey. V. anguillarum frequently been isolated from

North America, Pacific, North West of Europe such as Norway and Sweden (Patch and Kiehn,

1969), in Denmark and Atlantic Ocean (Larsen et al., 1994). Latin America is also threatened

with fish vibriosis caused by V. anguillarum (Rubio, et al., 2008).

V. anguillarum has been isolated from wide range of environments, including surface

water, water column, and sediments, from coastal waters and estuaries. It can be exist as free

living bacteria or associate with aquatic animal with or without any disease symptoms (Austin

and Austin, 1999). V. anguillarum may isolated from any part of the fish such as intestinal

tract, hemorrhagic ulcers, gill, mucus, spleen and kidney (Larsen et al., 1988). V. anguillarum

viii can be plated on any media sported with carbohydrate, nitrogen source and 1% to 3% of NaCl,

at neutral pH. Optimum temperature for cultivation is ranging between 20 ºC to 25 ºC, which

emerge as highly significant factor influencing the growth of this bacterium (Larsen, 1984). As

with other members of the genus Vibrio, this bacterium grows on selective media

Thiosulphate-citrate-bile salt-sucrose (TCBS) (Pfeffer and Oliver, 2003). Alsian et al., 1994

have developed Vibrio anguillarum medium (VAM) a medium for presumptive identification

of V. anguillarum. VAM is a marine agar supplemented with 1.5% NaCl, ampicillin and bile

salt.

2.1.5. Pathogenicity

Vibriosis is a stress related disease that associated with several factors like low oxygen

level and over-crowding. Water temperature and salinity fluctuation have great impact on fish

stress as well as bacterial activity with reports indicating the incidence of vibriosis to increase

significantly in the first half of summer season in temperate water regions (Hastein and Holt,

1972). Wild and farmed fish under stress, trauma or immune compromise condition become

susceptible to infection by V. anguillarum. The severity of the infection varies according to

bacterial strain, host species and environmental conditions, thus the symptoms may take the

form of hemorrhagic ulcers on mouth, skin, muscles, and fins; darkness is seen on the whole or

part of the fish, anorexia, and abdominal edema. Pale gills are usually noticed due to anemia.

Most often eye is infected resulting in gray corneal opacity or eye ulcer (Austin and Austin,

1993). Internally, spleen necrosis, fluid accumulation in the heart is noticed with development

of petechia tissue in liver and intestine. Histopathologically, the muscle fibers are widely

separated, with intramuscular connective tissue filling only some of the space, while the rest is

filled with fluid. An abundance of red blood cells and few leukocytes can be detected in the

intramuscular tissue (Ransom et al., 1984).

ix

In salmonids, although gastrointestinal tract serve as port of entry for vibrios, the main

symptoms observed are splenomegaly, liver and kidney necrosis, gill edema and occasionally

intestinal petechia (Horne and Baxendale, 1983). In a recent study, V. anguillarum infecting

rainbow trout was shown first to attach to skin, followed by penetration and biofilm formation.

The bacteria utilize skin mucus as chemoattractant, which may enhance the entry into the fish

host and latter the bacteria move to other internal organs such as spleen, liver, kidney, intestine

and blood (Croxatto et al., 2007). O’toole et al., 1999 have compared the mechanism of

chemotactic motility for both intestinal and skin mucus. Their study revealed that V.

anguillarum swims towards both types of mucus, with a noticeable higher chemotactic

response for intestinal mucus. Extensive chemical analysis demonstrates that intestinal mucus

contains considerably higher quantity of free amino acids and carbohydrates, which serve as

attractants.

2.1.6. Virulence factors

Pathogenic bacteria require several virulence factors to establish itself in the host and

cause the disease. Majority of the pathogenic microbes have multiple virulence factors that can

cause damage to the host and thus contribute to overall virulence phenotype of the microbe.

Virulence factors may include bacterial toxins, outer membrane proteins that mediate bacterial

attachment and hydrolytic enzymes that may add to the pathogenicity of the bacteria

(McClelland et al., 2006). Analysis the relatedness of the plasmid profiles and pathogenicity in

V. anguillarum showed the existence of correlation between virulence and the presence of a

50-megadalton plasmid (pJM1) (Crosa et al., 1977). The plasmid pJM1 is known to code for

iron sequestering proteins that enable the bacteria to obtain iron important for its metabolic

activity (Crosa, 1979). Resistance to the bactericidal mechanisms of serum appears to be an

important contributor in the virulence of V. anguillarum strains and shown to be not mediated

by plasmid (Trust et al 1981).

x

Quorum sensing, a type of cell to cell communication, coordinate bacterial activities in

the bacterial population, by using signal molecules. This communication is known to play a

fundamental role in regulation of virulence factors in pathogenic vibrios. The signal molecules

usually have low molecular weight and they are diffusible in nature e.g. N-acylhomoserine

lactones (Waters and Bassler, 2005). N-(3-hydroxyhexanoyl) homoserine lactone (3-hydroxy-

C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL) are the main quorum sensing signaling

molecules in V. anguillarum (Milton et al., 2001). This bacterium possesses three signal

receptor proteins, VanN, VanQ, and CqsS, these receptors channel the signal by

phosphorylation cascade dedicated for quorum sensing pathway (figure 2). At low density of

cells, in the absent of signaling molecules, the three receptors donate phosphoryl groups to

VanU, which lead to phoshphorylation of VanO and activate it. The activity of VanO leads to

expression of small regulatory RNAs which together with RNA chaperon Hfq destabilize the

central molecule of the quorum sensing pathway, VanT. At high density VanT, it induce the

expression of an extracellular proteases and positively regulates, pigment production, and

biofilm formation. In the stationary phase, the sigma factor RpoS stabilizes VanT through Hfq

suppression (figure 3) and links VanT to stress response in V. anguillarum (Weber et al.,

Milton, 2006; Weber et al., 2008).

xi

Figure 2. Model for quorum sensing in V. anguillarum (Weber et al., 2008).

Figure 3. Model for regulation of VanT expression (Weber et al., 2008).

xii

Secreted proteins have crucial effect on bacterial adaptation to the different

environments as well as in host invasion. V. anguillarum is similar to V. cholerae in utilizing

type 6 secretion system (T6SS), to secrete potent virulence factors such as, the two external

proteases EmpA, PrtV and the haemolysin-co-regulated-like (Hcp). T6SS is coded by partial

operon of eight genes VstA-H (figure 4). The proteins VstA-D show distinction from T6SS

proteins studied so far; whereas VstE-H are signature proteins associated with other bacterial

T6SS. The Hcp is down regulated by VstE-H and VstB, while VstA, VstC, and VstD. The

T6SS proteins also regulate expression of two extracellular proteases, EmpA and PrtV, but

inversely regulating Hcp expression. This regulation was indirect as T6SS positively regulated

expression of the stress-response regulator RpoS and the quorum sensing regulator VanT,

which positively regulate protease expression (Filloux, et al., 2008; Weber et al., 2008)

Figure4. T6SS gene clusters in vibrios (Filloux, et al., 2008)

T6SS gene clusters are associated with the icmF gene code for IcmF, which contains

transmembrane domains and a walker motif for binding nucleotides and plays a role during

survival in macrophages (Cascales, 2008).

2.1.6.1. Outer membrane proteins

Outer membrane of Gram negative bacteria consists of phospholipids and proteins. The

proteins make up to 50% of the outer membrane mass. Recently, these outer membrane

proteins (OMPs) have attracted attention, since they have important role in the host bacteria

interaction, adherence, uptake of nutrients including iron from the host, antimicrobial

resistance and subverting host defense mechanisms (Li et al., 2008).

xiii

Internalization plays an important role in V. anguillarum infection and survival in the

host. The strains which have a higher invasion capacity show a stronger attachment rate (Wang

et al., 1998). The adherence of the bacteria to the host cell is prerequisite for the

internalization. The mediator molecules of bacterial adherence to the cell surfaces are known

as adhesins. OMPs are the main adhesins in the bacterial cell (Wang and Leung, 2000). The

major OMP in V. anguillarum is outer membrane protein U (OmpU), 35-42 kDa characterized

by relatively weak cationic selectivity, moderate surface charge and classified under general

diffusion porin (Simón et al.,1996). OmpU plays role in bile salt resistant, which is considered

the first step in bacterial evolution to survive intestinal environmental condition and

subsequently bacterial colonization in the fish gut. (Wang et al, 2003). The ability of

osmoregulation is crucial to marine bacteria like V. anguillarum which always encounter

significant change in NaCl during the movement between sea water and host. The OMPs are

the main players in bacterial adaptation salinity fluctuation. In V. anguillarum, OmpU along

with OmpW are induced under salinity suggesting their role in NaCl efflux pump in contrast to

maltoporin which is suppressed under the same condition (Kao et al., 2009). The OMPs are

also involved in iron sequestering. OM2 is an OMP of 86 kDa is induced under iron limitation.

The gene responsible for coding this protein is present on the virulence plasmid pJM. (Actis et

al., 1985). Studies have emphasized the role of the OMPs in protective antigenicity as they are

highly immunogenic, with exposed epitopes on the cell surface that are conserved in different

serovars. Therefore OMPs considered ideal candidates in vaccine development (Koebnik et al.,

2000; Rahman and Kawai, 2000).

2.1.6.2. Siderophores

Most of the bacteria depend on iron for cell metabolism such as respiration, pigments

and energy production. Iron acquiring is one of the main challenges for marine bacteria due to

xiv limited sources of iron (Neilands, 1984). The challenge become more severe once the bacteria

have colonized inside the host body, since the presence of iron binding proteins transferrin in

serum or lactoferrin in secretions reduce the iron accessibility. Infectious bacteria require an

iron sequestering system to cope with iron limitation condition in-vivo. Several

microorganisms respond to iron limitation by synthesizing low molecular weight molecules

0.4–1.5 kDa with strong iron binding capabilities. These molecules termed as siderophores,

they are scavenge, solubilize and ultimately transport iron (III) inside the cell (Carvalho and

Fernandes, 2010; Weinberg, 1978). Pathogenic V. anguillarum strains have evolved competent

iron acquiring systems. The well known siderophore in V. anguillarum is anguibactin, a

catechol (o-diphenol) rather than monophenol molecule, with molecular weight of 0.35 kDa

(Actis et al., 1986). The ability of V. anguillarum sequester iron is concomitant with the

presence of the pathogenic plasmid pJM1, since it encodes for an efficient iron uptake system.

Alongside to anguibactin, this system consist of the outer membrane protein receptor OM2 a

86 kDa protein coded by the same pJM1 plasmid and OM3, a 79 kDa protein of chromosomal

origin (Crosa and Hodges, 1981; Walter et al., 1983). The OM2 86 kDa is an OMP encoded by

the fatA gene, while OM3 is dual 37 kDa integral membrane proteins encoded by the fatD and

fatC genes. The same operon consist fatB codes for 40 kDa cytoplasmic membrane embedded

lipoprotein that possesses periplasmic binding domains. The plasmid genes are negatively

controlled by the chromosome mediated FUR protein (Chen and Crosa, 1996; Chai et al.,

1998). V. anguillarum strains isolated from various geographical locations may harbor

plasmids that are highly related to pJM1 and that are also associated with the high virulence

phenotype of these strains. Plasmid pJHC1 is a pJMl like plasmid isolated from virulent strains

and encoded an iron uptake system that increase the level of the siderophore anguibactin

production. The genes responsible for increased anguibactin production are included within the

iron uptake region of plasmid pJHCl (Tolmasky et al., 1988). Lemos et al., (1988) have

xv reported a chromosomal mediated iron scavenging system in virulent strains of V. anguillarum

which lack the putative pathogenic plasmids.

2.1.6.3. Enzymes

The invasion of a host by a pathogen may require the production of bacterial

extracellular enzymes termed invasions. These enzymes act against the host by breaking down

primary or secondary defenses elements of the body or damaging the host cells thereby

enabling the bacteria to spread. The damage to the host as a result of this invasive activity may

become part of the pathology of an infectious disease (Hoew and Iglewski, 1984; Grey and

Kreger, 1979). Virulent strains of V. anguillarum produce a heat labile (70 ºC for 10 minutes)

external protein that is lethal to several fish. Further purification and characterization to this

exotoxin prove that it was a protease enzyme with a molecular weight of 36 kDa. This protease

is common in different serotypes of the bacteria. The LD50 of the external protease was about

1.7 microgram per gram of fish (Inamura et al., 1985). A mutant of V. anguillarum which

produced less amount of protease were 1000 times less able to cause infection by immersion,

which reflect a role of this enzyme during infection process (Norqvist et al., 1989). It was

observed that this enzyme required Zn2+ for its elastolytic activity and Ca2+ for its stability, and

therefore classified under metalloproteases. N-terminal amino acid sequence analysis of the V.

anguilarum protease revealed homology to Pseudomonas aeruginosa elastase ( Norqvist et

al.,1990; Milton et al., 1992). It has been proved that the gastrointestinal mucus induce empA

gene to produce the metalloprotese in V. anguillarum over 70 folds. The regulation of empA

gene was through quorum sensing molecules (Denkin and Nelson, 1999; Denkin and Nelson,

2004).

Bacterial hemolysins are extracellular virulent factors that have toxic effect on host

cells such as erythrocytes. Hemolysins are pore formers that create anion-permeable channels

xvi in cell membrane that subsequently cause ion leakage and, ultimately, cell lysis and cell death.

V. anguillarum produces at least five different hemolysins, vah1, vah2, vah3, vah 4, vah5. vah1

was the first studied hemolysin in V. anguillarum (Hieono et al., 1996) and Rodkhum et al,

2005 reported the four following hemolysin genes. The molecular size of VAH2, VAH3,

VAH4, VAH5 are 33, 75, 22, and 66 kDa respectively. Based on amino acid sequences,

hemolysin VAH2 shows the highest homology with V. vulnificus hemolysin, and VAH3,

VAH4 and VAH5 show the highest homology with V. cholerae hemolysins. Additionally, the

phylogenetic relationships of the V. anguillarum hemolysins show that they are all closely

related with V. cholerae hemolysins. These results suggest that probably V. anguillarum and V.

cholerae evolved from a common ancestor. All hemolysin mutants had lower virulence than

wild type V. anguillarum. The vah4 mutant was the weakest virulence among the other mutants

and thus it was suggested that vah4 gene is the strongest virulence gene among the four

hemolysin genes (Rodkhum et al, 2005).

2.1.7. Cultivation and identification of Vibrio anguillarum

V. anguillarum is a straightforward cultivated bacterium. It is able to grow on wide

range of selective and non selective media supplemented with 1.5% sodium chloride such as

trypticase soya agar (TSA) and brain heart infusion (BHI), or selective media such as

thiosulfate citrate bile sucrose agar (TCBS), marine agar (MA), V. anguillarum medium

(VAM) (Alsina et al., 1994 ). V.anguillarum is able to grow at an optimum incubation

temperature ranging between 15-25 º C and a pH between 5 and 9. As with most of the marine

vibrios V. anguillarum doesn’t grow in the absence of sodium chloride and requires a salt

concentration of 0.5-5.0%, the optimum being 1.0-3.0%. The colonies may be seen after 24,

48, or 72 hours of incubation (Larsen, 1984).

2.1.7.1. Identification by conventional methods

xvii

As described in the Bergey’s manual of Systematic Bacteriology (Baumann et al.,

1984), Vibrio anguillarum is a Gram-negative bacterium with short rods, may appear under the

microscope as curved, straight, or pleomorphic shape. The size of the bacterium is typically 0.5

to 0.8 μm in diameter and 1.4 to 2.6 μm in length. Vibrio anguillarum is a facultative anaerobe.

It is rapidly motile and has a β-haemolysin activity on blood agar. After 2 -3 days of culture on

agar, the cream colour colony appears with 2mm in diameter. Biochemically this bacterium is

catalase and oxidase positive. Amino acids utility test is considered a distinctive test of V.

anguillarum from most of marine vibrios since it can utilize arginine and unable to utilize

lysine or ornithine. It also produces indole and gelatinase. This bacteria reveal variation in

sugar fermentation, but generally the fish pathogen strains are sucrose and manitol

fermentative on the contrary unable to ferment inositol and melibiose (Pacha and Kiehn, 1969;

Pazos et al., 1993). The O/129 Vibriostatic test (2, 4-diamino-6, 7-di-iso-propylpteridine

phosphate) is the typical test which differentiates Vibrionaceae from Aeromonas

(Aeromonaceae members are resistant to O/129) but is complicated since other bacterial

species are also sensitive to this test such as Moritella, Photobacterium and Flavobacterium ,

in addition some V. anguillarum isolates are resistant to this test (Pedersen et al., 1995). This

species is easy to identify and distinguish from other Vibrio species (Alsina and Blanch, 1994a;

Alsina and Blanch, 1994b).

2.1.7.2. Identification by molecular techniques

Conventional methods for the identification and characterization of bacterial isolates

may fall short when a bacterium exhibit unusual phenotypic profile, beside the fact that they

require long time to peruse. Recent advances in DNA sequencing technology and molecular

biology techniques have greatly enhanced the ability to determine and identity bacterial

xviii isolates. DNA sequence information and growing availability of sequence databases are

significant movements developing the use of molecular methods for bacterial identification

(Kolbert and Persing, 1999). To compare bacterial genetic materials for the aim of

identification, genes which are present in all the bacteria such as rRNA genes are required. On

the other hand these genes should be highly conserved in the particular species. The copy

numbers, overall ribosomal operon sizes, nucleotide sequences and secondary structures of the

rRNA genes revealed that they are highly conserved within a bacterial species due to their

fundamental role in polypeptide synthesis. The 16S rRNA gene is the most conserved of the

rRNA genes therefore sequencing of this gene has been established as a universal standard for

identification and taxonomic classification of bacterial species (Bouchet et al., 2008).

The utilization of DNA-DNA hybridization technique has enabled bacterial

identification by synthesis of a labeled probe which is complementary to a unique sequence in

the 16S rRNA gene (Socransky et al, 1994). Comparative analysis of 16S rRNA sequences of

V. anguillarum and very closely related vibrios was enough to distinguish them into distinct

species (Wiik, et al., 1995). Rehnsatm et al. (1989) in their study designed a 25 bp RNA probe

used for specific identification of V. anguillarum. The detection limit of this procedure was

5000 cell per milliliter. Another probe were designed based on the updated vibrio 16S rRNA

sequences further enabled the specific identification of V. anguillarum chromosomal DNA

without any cross link (Picado et al 1994).

The application of polymerase chain reaction (PCR), sequencing, associated database

construction and searching software have created ample opportunities for microbial

identification. PCR is a technique, which uses a DNA polymerase enzyme to make a huge

number of copies of virtually any given piece of DNA or gene. It amplifies enough specific

copies enabling to carry out any number of other molecular biology applications. The target

sequence is identified by a specific pair of DNA primers, oligonucleotides usually about 20

xix nucleotides (Sparrt, 2004). PCR has used for identification of Vibrio anguillarum depending

on primers derived from rpoN gene. The detection prove to be highly sensitive that can detect

1 cell per reaction in case of pure culture of the bacteria, or 10-100 bacterial cells per reaction

in case of fish tissue contaminated samples, which corresponds to 2000 - 20000 bacterial cell

per gram of fish tissue. This technique is accurate and required 5 hours of work (Gonzales et

al, 2003). Another PCR based technique was developed based on the gene of N-

acetylmuramoyl-L-alanine amidase (amiB gene). PCR specificity was demonstrated by

successful amplification of DNA from V. anguillarum and by the absence of a PCR product

from 25 other Vibrio strains and various enteric bacteria. The PCR produced a 429-bp

amplified fragment from as little as 1pg of V. anguillarum DNA. The sensitivity of the

technique is limited to 10000 bacterial cells per gram of fish (Hong et al., 2007). These results

suggest that PCR technique using primers derived from the housekeeping genes considered as

to be a sensitive and species-specific detection method. Loop-mediated isothermal

amplification (LAMP) is an alternative method, like PCR, is based on DNA amplification. The

LAMP reaction is an auto cycling strand displacement DNA synthesis reaction using a DNA

polymerase with a high level of strand displacement activity and a set of specially designed

inner and outer primers. Four highly specific designed primers of LAMP are known to

hybridize simultaneously with six distinct sequence sites in the template DNA, while the PCR

primers hybridize with two distinct sequence sites. Therefore, DNA amplification in the

LAMP assay is more specific than PCR amplification. A successful trial has been made for V.

anguillarum identification based on LAMP using primers of empA gene. This method requires

1 hour of work, thus it is considered one of the fastest method for bacterial detection (Gao et

al., 2010).

Pulse Field Gel Electrophoresis (PFGE) is a technique which allows classification of

isolates into clusters. It relies on the digestion of DNA by restriction enzymes and subsequent

xx separation of the fragments by three directional gel electrophoresis using variable voltage. This

technique generated more profiles for V. anguillarum O1 than was found with ribotyping, and

therefore has a higher power of discrimination between isolates, making this technique a useful

tool for epidemiological studies (Pedersen et al., 1999).

2.1.8. Treatment

Before the era of vaccination, vibriosis was treated with antibiotics and antimicrobial

chemicals. The most common drugs used were tetracyclines, sulphonamides and quinolines

(Alcaide et al., 2005). Regardless of the efficiency of antibiotics in curing the infection, the use

of antibiotics in aquaculture is not straightforward. The first difficulty is drug administrated

which can be done either with food or in bath. The infected fish usually lose their appetite

hence it does not receive the adequate dose of treatment. The treatment of vibriosis by

antimicrobial chemicals like copper compounds cause harm to the fish and accumulate in the

tissues (Ellis, 1988). Another more significant risk comes from the development of resistance

to the antibiotic. Some reports revealed the emergence of V. anguillarum strains with multiple

antibiotic resistances, causing a significant threat to human health (Alcaide et al., 2005). In

recent years many investigations reporting a link between antibiotic use in food producing

animals emergence of antibiotic resistance in the bacteria of the treated animals and transfer

their resistance genes to human pathogens. The genetic markers of this resistance were located

on R plasmid (Aoki et al., 1974; Aoki et al., 1981). Additionally there is a consequently risk of

environmental contamination since the drugs are administrated to the entire ecosystem (Angulo

et al., 2004; Akinbowale et al., 2006).

Based on the reported risks and the increasing demand and expansion of aquaculture

there was an urgent need for the prophylactic treatment control bacterial infection in fish

including fish vibriosis.

xxi

2.2. Fish immune system

The immune system in finfish as well as other terrestrial vertebrate animals is able to

identify unique molecules in the intruders and develop specific immunity to cope with them.

This kind of immunity is gained its importance due to its persistence in the body for long

period (Ellis 1988; Secombes, 2008). Since the microorganisms’ infections can bring a

catastrophic results by killing the entire yield therefore the need of prevention is a must.

Vaccination emerge as important strategy for the success of large scale farming of commercial

fish such as, salmon, seabass, catfish, tilapia and cod (Sommerest et al., 2005).

The fish immune system involves a highly complex defense mechanisms utilizing a

wide range of structurally and functionally diverse tissues and organs, the principal function

being to protect fish from infectious agents and other toxic products. The system evolves wide

range of mechanisms to locate and eliminate foreign bodies. Immune system is often divided

into two subdivisions, nonspecific or innate immunity and specific or adaptive immunity; with

several boundaries define between the activities of these two types of immunity (Bowden,

2008; Harlow and Lane, 1988).

2.2.1. Innate immune system

The innate immune system is the sole defense system in lower animals such as

crustacean. In higher animals including finfish it is considered as the first line of defense. This

system is more active in finfish than other terrestrial counterparts (Savan and Sakai, 2006).

The innate system is further classified as humoral and cellular. Humoral immunity

includes complement, transferrins, anti-proteases, haemolysins, lysozymes, interferon and C-

xxii reactive proteins; while cellular immunity constitute of macrophages, neutrophils, lymphocytes

and scavenger endothelial cells systems. The innate immune system identifies the breach of the

body through non-specific signals such as LPS, peptidoglycans and β-glucans. The recognition

process involves receptor molecules such as C-reactive protein, lectin, mannose binding

protein or toll like receptors (Chakravarty, 2006). Complement system is one of the most

important players during pathogen infection. As compared to terrestrial animals, the fish

complement system is highly evolved, but more sensitive to temperature elevation. The

complement has a direct killing activity for the intruder or as an opsonisation molecule (Lange

et al., 2004). Cellular innate immune response starts with the neutrophils, which due to their

fast movement ability are able to reach first to the site of infection. Macrophages are also

involved in higher phagocytic activity, both types kill the intruder by producing hydrogen

peroxide (Ellis, 1999).

2.2.2. Adaptive immune system

Frequent exposure of vertebrate animals to a pathogen often result in surviving

individuals that resistant to subsequent infection to the same disease. The reason for such

adaptation is the adaptive immune system. The adaptive immune system is characterized by

the ability to elicit resistance to the pathogen based on recognition of specific antigen and

orienting of the whole immune system towards particular target. The system also involve

assemble of an immune memory to the subsequent encounter for the same antigen (Watts et al.,

2001).

The central theme of adaptive immunity lies in the lymphoid tissues. In fish, head and

kidney are the major lymphoid organs, while thymus and spleen are the minor lymphoid tissue

producers (Press and Evensen, 1999).

xxiii

When the intruder is first encountered in the host, an important phagocytic cell called

antigen presenting cell (APC) ingest it and display its epitopes on the surface. The epitope is a

peptide derived from the digested antigen and displayed on protein complex known as major

histocompatibility complex (MHC). There are two different types of MHC, MHC I expressed

on all nucleated fish cells (interacting with the CD8 molecule) and MHC II, expressed

exclusively on (APC), such as B cells, macrophages and on activated T cells, interacting with

CD4 molecules. A specific response requires the interaction of APC, B cells and T helper cells

as it shown in figure 5. Antigen presentation is a crucial part of the specific immune response.

In fish, the processing and subsequent presentation of the antigens is believed to be similar to

the processes described in mammals (Vallejo et al., 1992).

In response to an antigen, the B-lymphocyte produced gets activated by its

correspondent T-lymphocyte (Press and Evensen, 1999). However, unlike mammalian B cells,

fish are able to respond to non-specific inducers, without the involvement of T-cells (Li et al.,

2006).

The anamnestic response in fish is considered poor when compared to mammals,

possibly because immunoglobulin IgM is the main presenting antibody type, which shows

genetic diversity in terms of its ability to exist as a mono, dimmer or tetrameric form and being

highly flexible. A few other genes and immunoglobulin molecules such as IgD, IgT and IgZ,

are also reported to be involved, however their function is still not known (Danilova et al.,

2005 & Magnadottir, 1998).The environment such as temperature in which the fish has

evolved is influencing its specific immune response, affecting the production of an adequate

anamnestic response, especially regarding the T-dependent response (Bowdin, 2008).

xxiv

Figure 5. Response of adaptive immune system to the first encountered antigen by

(Iwama and Nakanishi, 1996).

2.3. Vaccination

The vaccine provides the host with resistance to a disease without previous exposure to

potential infection. Vaccine is considered as prophylaxis treatment, as it is used in preventing

infection. It works by enriching the specific immune system by desirable memory cells that

persist for long periods of time. The history of vaccine started in 1796, when Edward Jenner

demonstrated that humans could be protected against smallpox virus after an injection with a

live cowpox virus which known as vaccinia. The term vaccine was then coined by Jenner

himself in 1798. Therefore vaccine can be defined as a biological preparation that improves

immunity to a particular disease. A vaccine is made from attenuated or killed forms of the

microbe or its subunit in a way mimicking the natural infection. The key factors of a successful

vaccine are specificity and memory; also it should be safe without any side effect on the host

xxv and effective so that it provide enough protection to the vaccinated animal (Ellis, 1988; Stern

and Markel, 2005).

2.3.1. Fish vaccination

Snieszko and colleagues in 1938 were the first to demonstrate the ability of carps to

develop an anamnesis resistance, when injected with heat killed Aeromonas punctata. Duff in

1942 proved that oral immunization could successfully protect fish against A. salmonicida

using killed bacteria. Fish vaccination development was affected with the development of

antibiotics in 1940s. However, the appearance of antibiotic resistant bacteria in 1970’s

rematerialized the need of fish vaccine development (Ellis, 1988 &Van Muiswinkel, 2008).

Vaccine development starts with identification of suitable antigens which could be recognized

by the fish immune system, and the induction of the vaccine in the optimum form and route, so

that the animal develops a strong protective immunity (Gudding et al., 1999). The vaccine

types used in aquaculture are classified into three categories; first is whole pathogen, second

subunit and recently DNA vaccines (Munn, 1994).

2.3.2. Whole cell vaccine

The whole pathogen vaccine can either be a killed vaccine or live attenuated vaccine.

Killed vaccine may lack important de novo proteins induced upon the infection, such as

secreted toxins which could potentially be protective antigen; therefore it is considered a

substandard to the live attenuated whole bacterial vaccine (Hastein et al., 2005). A comparison

between V. anguillarum formalin killed vaccine against membrane extract revealed that the

formalin killed bacteria is more protective than the membrane extract in the intraperitoneal and

oral administration. The contrast was seen when both the vaccine preparations were feed orally

to the fish (Agius et al., 1983).

xxvi

Attenuated vaccines are still rare in aquaculture due to the possibility of reversion of the

attenuated bacteria to its potentially pathogenic nature. Nevertheless a lot of research is taking

place with some promising results for many pathogens such as Streptococcus iniae and

Aeromonas hydrophila. The reversions are valid danger, especially since the pathogen could be

potentially shed from the infected animal into the aquatic environment (Locke et al., 2008;

Vivas et al., 2004). Further, the preparation of multivalent vaccine with whole killed pathogens

can lead to the inhibition of the specific response expected the individual pathogen induced

protection, due to induction of suppression immune system. Several vaccines formulated with

inactivated pathogens have failed to protect fish against bacterial and viral diseases (Romalde

et al., 2005). Avirulent mutant of V. anguillarum has been utilized to prepare live attenuated

vaccines against heterologous strain of V. anguillarum. Protective immunity was recorded one

week after bath vaccination for 30 min with 1010 bacteria per liter of water. A single-dose

immunization was effective for at least 12 weeks. The presence of common antigenic

determinant with Aeromonas strain has made this vaccine a successful multivalent vaccine

(Norqvist et al., 1989).

2.3.3. Subunit vaccine

Subunit vaccines are based on the production of a particular antigen either purified

directly from culture of the pathogen, expressed by a prokaryotic or eukaryotic system or even

chemically synthesized peptide vaccines. The main advantage of subunit vaccine is the absence

of pathogen-derived toxins or immunosuppressive components. The subunit vaccines are

preferably parts of pathogen surface antigens such as outer membrane proteins due to their

accessibility to the host immune system (Munn, 1994). The subunit vaccine can be produced

by recombinant technology. The advantage of recombinant vaccine is that there is no chance of

host being threatened by vaccine material. Moreover, each batch of vaccine has the same

potency and is more stable during the long term storage, it also helps to remove the bacterial

xxvii antigens or cleave the epitopes which stimulate T-suppressor cells (Lorenzen, 1999). The main

difficulties of the development of a subunit vaccine are the selection of the antigen molecule

and then expressing the target molecule in its desired 3D structure (Grandi, 2001). The

recombinant protein technology has already been used to produce OmpW, OmpV, OmpK and

OmpU successfully for the aim of vaccine development against V. parahaemolyticus in large

yellow croaker (Pseudosciaena crocea) by intraperitoneal injection (Mao et al., 2007). Outer

membrane protein K of V. alginolyticus used to increase the specific protectively in fish. This

study showed that the conserved OmpK was an effective vaccine candidate against infection

by V. alginolyticus (Qian et al., 2008). Li, et al. (2008) has indicated that the OmpK of Vibrio

harveyi is an effective vaccine candidate against homologues bacterial infection in Orange-

spotted groupers. Outer membrane proteins are widely tested as subunit vaccine since they are

highly immunogenic bacterial components due to their exposed epitopes on the cell surface,

more over unlike toxins and most of the enzymes, the outer membrane protein lack the toxic

effect on the host (Lorenzen, 1999).

2.3.1.3. DNA vaccine

DNA vaccine is an in situ produced vaccine. The principle is based on the injection of a

plasmid coding a protective antigen, letting the host machinery transcribe and translate the

chosen gene (Liu, 2003). Therefore DNA vaccine is considered stable, additionally several

protective genes can be theoretically injected in different plasmids, thus DNA vaccine is one of

the most applicable approach for fish protection (Gurunathan et al., 2000). Historically the first

DNA vaccine approved in the US was for horse West Nile virus in 2005 and the second was

for fish (Infectious Haematopoietic Necrosis Virus DNA vaccine, Novartis), but the hurdle of

licensing this vaccine was quite difficult since the fish were farmed for human consumption.

The main objection for the application of fish DNA vaccine is the possibility, that the designed

plasmid to be integrated into the host’s genome, and subsequently distributed in other

xxviii organism including humans when it is released into the environment (Rogan and Babiuk,

2005). However this possibility has been reported to be extremely unlikely (Lorenzen and

LaPatra, 2005). A preparation of a V. anguillarum major OMP DNA vaccine has shown a

moderate level of protection against V. anguillarum in Asian seabass fish immunized with a

single intramuscular injection. Antibodies against the chosen protein were detected even after

seven weeks from the vaccination (Kumar et al., 2007). Recently, a number of new techniques

have been developed to introduce a foreign DNA into cells such as chitosan encapsulation

which allow oral delivery for the vaccine molecules. V. anguillarum Major OMP DNA vaccine

preparation has been used in oral administrated vaccine using chitosan nanoparticles

encapsulation. However a successful expression of the selected gene in vivo in the fish’s

tissues was detected but the vaccination revealed a 46% of protection against V. anguillarum

(Kumar et al., 2008).

2.3.5. Strategy of vaccination

Most of the antigens fail in inducing strong immunity response due to the lack of proper

mimic to natural infection in persistence or when the antigen are ignored by immune system.

Therefore adjuvants are emerged as solution for such problems, since they help in increasing

the immune response of the immunogenic molecules (Harlow and Lane, 1988). These

substances either increase the antigen uptake and cell presentation or allow prolonged

persistence of antigens (Evensen et al., 2005). They also may activate the lymphocyte in a

signaling manner (Secombes, 2008).The most common adjuvant is freund’s mineral oil, it was

shown that it prolongs the persistence of the vaccine molecule for more than a year

(Chakravarty, 2006). Many adjutants are used in fish vaccines to avoid oil formula (which

seems to produce the best results) such as glucans and aluminium salts (Midtlyng, 1996). The

common side effect of the adjuvant include reduced growth rate, local reactions with the

xxix appearance of granulomas or organ adhesion in the peritoneal cavity depending on the fish and

oil formula (Poppe and Breck, 1997; Midtlyng et al., 1998; Midtlyng and Lillehaug, 1998).

Numerous vaccine delivery methods are available to the fish farmer. The widespread

vaccine administration method been adopted is the intraperitoneal injection followed by

immersion and finally oral vaccination (Palm et al., 1998). Injection permits the use of

adjuvant, but results in high level of stress for the fish since it require anaesthetization and it is

impossible to pursue to a fish below 15 grams (Hong et al., 2009; Vandenberg, 2004). Injection

route has been used to deliver OmpK of V. alginolyticus and V. harveyi as subunit vaccine

(Qian et al., 2008; Zhang et al., 2007). Practically oral vaccines are easier to deliver than other

treatments particularly for large scale farming, since it doesn’t interfere with general farm

activities, doesn’t stress the fish and can be tried on any fish size; however it shows poor level

of protection. The major problem is the destruction of the antigens before reaching the lower

gut (Ellis, 1988). Methods to protect the antigens from degradation are required, such as

encapsulation or gastric section neutralization (Azad et al., 2000; Maurice et al., 2004). Kumar

et al., (2008) has used the oral administration for delivery of DNA vaccine to develop a

moderate protection for Asian seabass against V. anguillarum.

Immersion vaccination is carried out by dipping the fish in vaccine solution for a period

of time. The main portal of the antigens in this technique is the gills tissue (Smith, 1982).

Vaccination of rainbow trout with 1010 cell/liter attenuated V. anguillarum through bath

vaccination 30 min secured protective immunity for a period ranged between 1-12 weeks

(Norqvist et al, 1989). Subunit vaccination can also be carried out via immersion like in case of

OmpW of V. alginolyticus (Qian et al., 2008).

2.4. Bioinformatics

xxx

Bioinformatics is the science which deals in collecting, processing and analysis of

molecular biology information with the aim of a better understanding, significant of data and

further expanding of biological data. While genomics deals with storage and analysis of DNA

sequences proteomics is involved in studying the nature and function of the proteins of interest

using computational softwares (Benton, 1996).

Vast amount of DNA sequences have been determined and new sequences are

characterized in continuous acceleration. Computers are necessary to store and distribute this

huge volume of data. The principle data banks where these sequences are stored are the

GenBank at the National Institute of Health, Bethesda, Maryland; EMBL (European Molecular

Biology Laboratory) sequence data base at the European Bioinformatics Institute, Heidelberg

and the DDBJ (DNA Data Bank) of Japan. These databases continuously exchange newly

reported sequences and make them available to molecular biologists throughout the world via

the internet (Akash, 2007).

2.4.1. Sequence analysis tools

Bioinformatics play an important role in gene identification, gene finding, genotyping

and genetic analysis. Public sources of databases and tools abound, although it is sometimes

difficult to determine the quality, consistency and sustainability of these sources.

Bioinformatics data integration and tool standardization are critical to the success of

association and linkage studies. The underlying data models accommodate the variability

inherent in subject collections, the ability to trace the data source, and the automation and

archival storage of the analyzed results (Rao et al., 2009). The bioinformatics applications

involved in this study are briefly described as follows.

2.4.1.1. Sequence alignment

xxxi

Genbank provides enormous number of gene and protein sequences. The DNA and

protein sequences deposited in this database present an opportunity to identify genes and

predict the nature and function of the translated protein by comparing the gene of interest with

similar previously studied genes (KILEY, 1992). The BLAST (Basic Local Alignment

Search Tool) is widely used for searching protein and DNA databases for sequence

similarities. BLAST program have been designed to compare protein or DNA queries with

protein or DNA databases in any combination, (Altschul et al., 1997). To analyze the BLAST

algorithm and its refinements, the statistics of high-scoring local alignments are required.

Optimal alignments are called high-scoring segment pairs (HSPs) (Karlin and Altschul, 1990).

The advantage of local alignment is that proteins with a common domain can be identified.

Significant statistical results, allow the prediction of function of the target protein and even the

possible structure (Jain, 2002). Further information on consensus sequences and protein motifs

can be extracted via multiple alignments. Multiple alignments are also used to find

evolutionary relationships between the homologes. This approach also provides a clue into the

structural features of the protein (Fischer and Eisenberg, 1999).

2.4.1.2. Signal peptide prediction

Signal peptides (SPs) are short regions guide secretory proteins to the correct

subcellular sites in the cell. Generally they are positively charged segments known to play a

role in the proper orientation of the protein in the lipid bilayer. They are cleaved off by a signal

peptidase upon reaching protein destination (Choo et al., 2009). Prokaryotic and eukaryotic

cells utilize these short peptides to mediate translocation of the passenger protein domains

across the endoplasmic reticulum membrane in eukaryotes or the inner and outer membranes in

prokaryotes (Spiess, 1995). In vivo task involves the identification of these SPs and the correct

identification of their cleavage sites for subsequent production of the mature protein sequences.

Bioinformatics approach compares the unprocessed sequences in the sequence databases with

xxxii results obtained by experimental methods. This has catalyzed the development of faster and

more accurate computational methods to automate the task of SP prediction (Choo et al.,

2009). SP prediction is fundamentally important as it impacts on other features such as

transmembrane topology, structure modeling, prediction (Kanagasabai et al., 2007) and

subcellular localization assignment proteins (Boden and Hawkins, 2007). The signal peptide

constitutes the N-terminal part of the protein (Gierasch, 1989; Lee and Bernstein, 2001).

There are a number of computational methods for identifying signal peptides and their

cleavage sites from the sequence of proteins. SignalP is the most common server used for

signal peptide prediction (http://www.cbs.dtu.dk/services/SignalP/). It exploits combination of

several artificial networks (Nielsen et al., 1997). The signal peptides from gram-positive

bacteria are significantly longer than those from other organisms. The average eukaryotic

signal peptide is 22 amino acids; in case of the gram-negative it is 25 amino acids while the

average gram-positive signal peptide contains 32 amino acids (Von Heijne and Abrahmsen,

1989).

2.4.1.3. Protein model prediction

Protein structures have proven to be a crucial portion of information for biomedical

research. Of the millions of currently sequenced proteins only a small fraction is

experimentally solved for structure determines. In the late 1950s Anfinsen and co-workers

suggested that an amino acid sequence carries all the information needed to guide protein

folding into a specific shape of a protein. Currently, there are more than 6,800,000 protein

sequences accumulated in the non-redundant protein sequence database (NR; accessible

through the National Center for Biotechnology Information:

ftp://ftp.ncbi.nlm.nih.gov/blast/db/) and fewer than 50,000 protein structures in the Protein

Data Bank (PDB; http://www.rcsb.org/pdb/). With these numbers at hand, it seems that the

xxxiii only way to bridge the growing gap between protein sequences and structures is computational

structure modeling (Kryshtafovych and Fidelis, 2009). Though membrane proteins represents

roughly about one-third of the proteins encoded in the genome. So far less than 1% of the

proteins structure is known. This reflects the difficulties of performing structural studies on

proteins of this type. As membrane proteins are amphiphilic containing both water soluble and

hydrophobic components, they associate the same difficulty with handling of proteins as they

are considered tricky to handle biochemically and difficult to overexpress by the cell. Apart

from these technical hurdles, structure determination is highly cost intensive. Therefore an

alternative approach for structure determination is in silico bioinformatics based approach,

which gives reasonably good 3D structure of proteins depending on the extent of similarity

with experimentally determined 3D structure of protein in the database (Spencer et al., 1999;

Bansal, 2005).

Globular protein domains are typically composed of the two basic secondary structure

types, the alpha-helix and the beta-strand, which are easily distinguishable. Other types of

secondary structures such as turns, bends, bridges and non-alpha helices are less frequent and

more difficult to be observed. The non-alpha, non-beta structures are often referred to as coil or

loop and the majority of secondary structure prediction methods are aimed at predicting only

these three classes of local structure. Given the observed distribution of the three states in

globular proteins (about 30 % alpha -helix, 20 % beta- strand and 50 % coil), random

prediction should yield about 40 % accuracy per residue. Modern secondary structure

prediction methods have crossed a sustained level of 76 % accuracy (Koh et al., 2003). The

secondary structure prediction methods typically perform analyses not for the single target

sequences, but rather utilize the evolutionary information derived from multiple sequence

alignment provided by the user or generated by an internal routine for database searches and

alignment (Levin et al., 1993). Membrane proteins are associated with the cell membrane and

comprise at least one transmembrane segment. Due to the hydrophobic environment within the

xxxiv cell membrane, the transmembrane segments are generally hydrophobic. Additionally typical

cytoplasmic membrane proteins comprise hydrophobic alpha-helix regions separated by

hydrophilic loops. On the other hand, bacterial outer membrane proteins exhibit a

characteristic beta-barrel structure comprising different even numbers of beta-strands.

Therefore specialized structure predictors have been designed for both types of membrane

proteins. Since both sides of the lipid bilayer are nonequivalent, structure prediction methods

for transmembrane proteins often attempt to identify not only the secondary structure elements,

but also the topology of the protein, such as the orientation of the elements with respect to both

surfaces (Von Heijne, 1992). Currently PRED-TMBB is widely trusted program for prediction

of transmembrane segments and topology. The PRED-TMBB

(http://bioinformatics.biol.uoa.gr/PRED-TMBB) is capable of predicting the transmembrane

strands and the topology of beta-barrel outer membrane proteins of Gram-negative bacteria.

The server reports the predicted topology of a given protein and score value that indicate the

probability of the protein being an outer membrane β-barrel protein. (Bagos et al., 2004).

3D structure prediction provide valuable insights into the molecular basis of protein

function allowing an effective design of experiments, such as site-directed mutagenesis, studies

of disease-related mutations or the structure based design of specific inhibitors. However, the

prediction of 3D structure from amino acid sequence is limited (Wolf et al., 2000). Successful

model building requires at least one experimentally solved 3D structure (template) that has a

significant amino acid sequence similarity to the target protein. The growing number of

structural templates brings a steadily increasing number of sequences into modeling distance

for comparative modeling. The core of the model is generated by recognition of the backbone

atom positions of the template structure. Thus templates are weighted by their sequence

similarity to the target sequence, while significantly deviating atom positions are excluded;

finally best model selected using a scoring scheme, which accounts for force field energy,

xxxv steric hindrance and favorable interactions like hydrogen bond formation (Brenner, 2001).

SWISS-MODEL (http://swissmodel.expasy.org/) represents a server that gives an opportunity

to generate preliminary three-dimensional models of the target structure based on the

alignments proposed by all servers. These models may be incomplete and contain significant

errors even if they are based on correct templates, but usually serve as a useful starting point

for further refinement. SWISS-MODEL is continuously developed to improve the successful

implementation of expert knowledge into an easy way to use homology modeling (Schwede, et

al., 2003).

2.4.1.4. Antigenic determinants prediction

Epitopes or antigenic determinants are antigen regions that are recognized by the

lymphoid cells. Antibody is synthesized with a part that recognizes the epitope, known as

paratope. Thus the prediction of protein antigenic structures is important for vaccine

development and drug evaluation, but is a time consuming task. Experimentally, to accurate

outline antigenic determinants, preparation of large number of well characterized peptide

fragments from the original protein is required followed by testing these derivatives for

immunological activity (Atassi and Lee, 1978). Alternatively, a homologous series of proteins

may be used to assess the influence of particular amino acid substitutions, thereby implicating

certain regions as antigenic determinants. This approach requires knowledge of complete

primary structures for a number of proteins before the immunological results can be interpreted

(Jemmerson and Margoliash, 1979). Hopp and Woods (1981) developed a bioinformatics

based method for locating protein antigenic determinants by analyzing amino acid sequences

in order to find the point of greatest local hydrophilicity. This was accomplished by assigning

each amino acid a numerical value (hydrophilicity value) and then repetitively averaging these

values along the peptide chain. The point of highest local average hydrophilicity is invariably

located, or the immediately adjacent region, as an antigenic determinant. It was found that the

xxxvi prediction success rate depended on averaging group length with hexapeptide averages

yielding optimal results. This method was developed using 12 proteins for which extensive

immunochemical analysis has been carried out. As more information becomes available on

protein antigens, it should be possible to use this information to predict the locations of

antigenic determinants before any immunological testing has been carried out.

B-cell epitopes prediction was the aim for many bioinformatics investigation since these

epitopes are sites of the molecules that are recognized by antibodies of the immune system.

Knowledge of B-cell epitopes is used to design of vaccines and diagnostics test kits

(Schellekens et al, 2000). The best single method for predicting linear B-cell epitopes so far is

the hidden Markov model. Combining the hidden Markov model with one of the propensity

scale method, Larsen et al., (2006) developed the BepiPred method. This method tested on the

validation data and proved to be significantly better than any of the other methods tested.

BepiPred server is publicly available at http://www.cbs.dtu.dk/services/BepiPred.

2.4.2. Phylogenetic analysis

Phylogenetic analysis is the process used to determine the genetic evolutionary

connections between species. The results of an analysis can be drawn in a hierarchical diagram

called a cladogram or phylogram (phylogenetic tree). The branches in a tree are based on the

hypothesized evolutionary relationships (phylogeny) between organisms. Each member in a

branch, also known as a monophyletic group, is assumed to be a descendent from a common

ancestor. Although originally, phylogenetic trees were created using morphological

characteristics, the development of molecular biology has provide a molecular mean for

determination the evolutionary relationships through matching patterns in nucleic acid and

protein sequences During evolution, it is very common for a gene to be duplicated. The copies

xxxvii continue to evolve separately, resulting in at least two similar instances of the same gene along

the genome of a species. (Saitou and Nei, 1987).

Phylogenetic analysis also gives the evidence how the pathogenic bacterium evolved.

Modern phylogeny methods information extracted from mainly DNA and protein sequences

after aligning several of these sequences and using only blocks which were conserved in all the

examined species. (Martinez-Murcia et al., 1992)