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ADONAY, JESSA C., Bachelor of Science in Biology, Sciences Cluster, University of the
Philippines - Cebu, Lahug Cebu City, Philippines, June 2014, QUANTITATIVE
EXPRESSION OF PISCIDIN-2, PISCIDIN-3 AND HEPCIDIN IN NILE TILAPIA
(Oreochromis niloticus) IMMUNIZED AGAINTS AEROMONADS PATHOGENS

Adviser: Marcos B. Valdez, Jr., Doc. Agr. Sci.

Co-Advisers: Apolinario V. Yambot, PhD, Dr. Yasser C. Cabansag, Paula Blanca Gaban

I. INTRODUCTION

1.1 Rationale

All populations, whether terrestrial or aquatic, are partially or completely limited by

occurrence of diseases, (Real, 1996). These diseases are caused by pathogens, such as

viruses, bacteria and fungi. The presence of these pathogens triggers the activation of bodily

responses, more specifically, the immune system. Immune-related genes play an important

role by coding proteins that either eliminate these foreign pathogens or catalyze the

elimination of the said pathogens, and consequently maintain homeostasis. (Pellitero, 2008).

Among the economically important populations that are periodically limited by

diseases are the aquaculture environments. Aquacultures cater to the demands of various

aquatic food resources, (Boyd, 2004). Tilapia, for instance, is important economically, being

the eighth most cultured group of fish worldwide, (Mjoun, Rosentrater, & Brown, 2010)

Aquacultures of tilapia are mainly utilized for food production, breeding and aquatic weed

control. (Boyd, 2004). Currently, the most cultured and popular tilapia species is

Oreochromis niloticus or Nile tilapia. It is the fifth most cultured species worldwide,

representing 84% of total global tilapia production, (Mjoun, Rosentrater, & Brown, 2010). In

the Philippines, it is the third most important fish cultured in terms of value and volume,

(Claus, 2013) and (Bureau of Fisheries and Aquatic Resources, 2010).

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Decline in tilapia production is usually caused by an outbreak of disease, which is

often associated with incidence of fish kills, (Fishbase Organization, 2012). One of the most

common groups of tilapia pathogens are the Aeromonads. Aeromonas hydrophila cause

Motile Aeromonas Septicemia (MAS), septicemia, ascitis, erosion, ulceration, detachment of

scale, exophthalmia and organ lesions to various fish species (Ibrahem, Mostafa, Arab, &

Rezk, 2008). Other Aeromonads such as A. caviae and A. veronii biotype sobria also cause

septicemia and ulceration, (Shayo, Mwita, & Hosea, 2012). In the Philippines, outbreaks

caused by members of the genus have been recorded in low volume and high density

aquacultures, especially in cage cultures since the early 1990s, (Yambot, 1998).

The pursuit for higher production of disease-resistant O. niloticus, as well as other

aquaculture organisms, has encouraged studies on the development of fish vaccines, as well

as, immunostimulants and targeted-drug formulations, (Maqsood, Singh, Samoon, & Munir,

2011). In the Philippines, substantial and continuous programs concerning aquaculture

research and extension have been done since 1972, (NFTTC, BFAR and FAC-CLSU on

Asian Development Bank, 2005). To date, molecular methodologies have gained popularity

in aquaculture research, (Sahoo & Saurabh, 2008). Analysis of gene-expression for instance,

have provided basis in vaccine development, among others.

Gene expression is the fundamental level at which the genotype gives rise to a

phenotype. There are several methods used in quantifying gene-expression, although Real-

Time PCR is the most preferred as it is sensitive, efficient and only requires low amount of

sample, (Gause & Adamovicz, 1994).

The best method to prevent infection against Aeromonas infection is to never have the

disease, (Pridgeon, Zheng, & Klesius, 2010). In this study, O. niloticus fingerlings and grow-

outs are to be vaccinated by immersion and injection method, respectively, against the
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Aeromonas pathogens, and the expression of their Piscidin-2, Piscidin-3 and Hepcidin genes

is to be quantified using RT-PCR. Piscidin-2, Piscidin-3 and Hepcidin, are among the

antimicrobial peptides (AMPs) in O. niloticus, that are associated with innate immunity,

(Peng, Lee, Hour, Pan, Lee, & Chen, 2012); (Robertson, 2011).

Expression of the said genes in fish when challenged with various fish pathogens has

been previously studied. Furthermore, their general role when exposed to pathogens has also

been elaborated (Colorni, Ullal, Heinisch, & Noga, 2008; Peng, Lee, Hour, Pan, Lee, &

Chen, 2012; Robertson, 2011). However, the quantification of the expression of O. niloticus’

Piscidin-2, Piscidin-3 and Hepcidin when vaccinated against A. hydrophila, A. caviae and A.

veronii, is not yet established. This study may provide additional information on the AMPs’

role and/or mechanisms in O. niloticus immunity and deliver baseline information to aid

actual vaccine formulation and the development of other aquaculture innovations, in support

for the economic goal of producing disease-resistant strains, (Poonsawat, Areechon,

Srisapoome, Maita, & Endo, 2009).

1.2 Objectives

The general aim of this study is to quantify Hepcidin, Piscidin-2 and Piscidin-3

expression in Oreochromis niloticus fingerlings and grow-outs, when immunized against the

Aeromonas pathogens namely, A. hydrophila, A. sobria and A. caviae. Specifically, it aims to

establish trends of gene-expression prior to vaccination and during the 14th, 21st, and 24th day

of post-vaccination and infection; and further, compare gene-expression of Aeromonad-

immunized to that of unimmunized O. niloticus individuals.

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1.3 Scope and Limitations of the Study

This study is only limited to the quantification of Piscidin-2, Piscidin-3 and Hepcidin

in O. niloticus fingerlings and grow-outs vaccinated by immersion and injection, respectively,

against Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii biotype sobria. In

addition, the expression of the said genes shall be quantified using Real-time PCR. This study

still does not concern on formulating market-quality vaccine against Aeromonas, but may

serve as a guide for such.

1.4 Significance of the Study

The quantification of gene expression may provide baseline information on vaccine

development against the pathogenic Aeromonads, which may further accommodate the

possibility of producing Aeromonad-resistant O. niloticus individuals, thereby, increasing

economic benefits.

1.5 Time and Place of Study

This study will be conducted from June to August 2014 at the Molecular Biology and

Biotechnology Laboratory of the College of Fisheries in Central Luzon State University,

Science City of Muñoz, Nueva Ecija.

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II. REVIEW OF RELATED LITERATURE

2.1 Tilapia Industry

Tilapia is one of the most important groups of aquaculture fishes globally, being the

eighth in terms of weight of production. In 2007, the commercial production of tilapia was

2.5 million tons, with a market value of $3.3 billion, (FAO, 2009 on Mjoun, Rosentrater, &

Brown, 2010). The species Oreochromis niloticus (Nile Tilapia) alone, ranked fifth among

the most cultured species globally, following silver carp, grass carp, Japanese carpet shell

(small-neck clam), and common carp, respectively. It contributed about 83% of total tilapia

production, with overall production of 2.3 tons, (FAO, 2009 on Mjoun, Rosentrater, &

Brown, 2010).

Tilapia cultures have been practiced since the ancient Egyptian times, as depicted on

an Egyptian tomb which dates back over 4000 years. However, significant worldwide

distribution of tilapia occurred during the 1940s and 1950s, with which the less desired O.

mossambicus was primarily distributed. O. niloticus or Nile tilapia was only distributed

during the 1960s and 1980s. It was introduced to the Philippines from Thailand in the 1960s,

(FAO, 2014). Currently, the main producers of tilapia are China (1.13 million tons), Egypt

(290,000 tons), Indonesia (206,000 tons), the Philippines (241,000 tons), Thailand(180,000

tons), and Brazil (100,000 tons), (FAO, 2010 on Mjoun, Rosentrater, & Brown, 2010).

In the Philippines, Tilapia is the third most important fish cultured, next to seaweed

and milkfish, in terms of volume, and after milkfish and tiger prawn, in terms of value,

(Claus, 2013 and Bureau of Fisheries and Aquatic Resources, 2010). The consistent major

production areas in the Philippines are Regions III (Central Luzon) and IV (Southern

Tagalog), (Asian Development Bank, 2005). Substantial and continuous programs on

aquaculture research and extension have been done since 1972, to cater to the increased
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demand for tilapia production. These programs were especially concerned on formulation and

dissemination of improved breeds, (NFTTC, BFAR and FAC-CLSU on Asian Development

Bank, 2005).

The International Center of Living Aquatic Resources Management (ICLARM),

Manila, for instance, helped incorporate genetics and molecular techniques to improve

performance of Nile Tilapia breeding, as this was not done until the mid-1980's, (Pullin &

Capilli, 1988 on Asian Development Bank, 2005). Donations from private institutions have

also aided the funding for improved farmed tilapia breeds and distribution, allowing farmers

to have access to improved tilapias strains, (Asian Development Bank, 2005). Valuable

strains available in the market include: FaST (FAC Selected strain), GMT (Genetically Male

Tilapia), GIFT (Genetically Improved Farmed Tilapia) and GET-Excel (Genetically

Enhanced Tilapia), (Claus, 2013). These innovations have led to the increase in production,

which consequently led to the decline of tilapia price, benefiting the consumers, (Garcia 2011

on Claus, 2013).

Similar with most organisms, O. niloticus encounters its own parasites which cause

the decline of its culture and production. The species are especially vulnerable as they are

usually cultured in high densities. High stocking densities evident in most aquacultures, led to

outbreaks of parasites and diseases, especially those with non-optimal hatchery design and

management. In such instances, fish kills were evident, which subsequently decreased

production and income, (Yambot, 1998 and Fishbase Organization, 2012). In addition, some

parasites which affected the species also affected other freshwater finfishes, (Fishbase

Organization, 2012).

Tilapia became a common component of Filipino and western diet as it has high

nutritional value, mild taste. It is also relatively less expensive compared to other finfishes,
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(Mjoun, Rosentrater, & Brown, 2010 and Asian Development Bank, 2005). In fact, tilapia

has replaced galunggong as 'poor man's fish', (Claus, 2013). It is rich in protein, phosphorus,

potassium, selenium, niacin, and vitamin B-12. In addition, it is low in fat and saturated fat,

omega-3 fatty acids, calories, carbohydrates, and sodium, (Mjoun, Rosentrater, & Brown,

2010).

2.2 Oreochromis niloticus

2.2.1 Taxonomic Classification and Etymology

Kingdom Animalia
Phylum Chordata
Class Actinopterygii (ray-finned fishes)
Order Perciformes (Perch-like)
Family Cichlidae (Pseudocrenilabrinae)
Genus Oreochromis
Species O. niloticus (Linnaeus, 1758)
Etymology Oreochromis: Latin, aurum = gold + Greek, chromis= fish/perch

niloticus: From Nile River

2.2.2 Physical Characteristics

Nile tilapia (Figure 2.1) is a deep-bodied fish with cycloid scales. It is silver in color

with olive/grey/black body bars present throughout the truncated caudal fin. The average size

at maturity ranges from 6-28cm, and weighs up to 60g. Its body is compressed with caudal

peduncle depth equal to its length. Protuberance on the dorsal surface of its snout is absent.

Similar with the members of the Chichilid family, its lateral line is interrupted. Its dorsal fin

has continuous spinous and soft rays. The species does not exhibit sexual dimorphism. The

pectoral, dorsal and caudal fins usually flush red during breeding season. In aquacultures

they usually attain sexual maturity at the age of 5-6 months. The species can live longer than
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10 years. However, they are highly limited by food availability and water temperature, (FAO,

2014; Fishbase Organization, 2012).

Figure 2.1 Oreochromis Niloticus (Nile Tilapia)

Source: S. Laurie-Bourque , ©Canadian Museum of Nature (top); ©Sustainable Sushi (bottom)

2.2.3 Habitat

O. niloticus is native to central and North Africa and the Middle East. It is a tropical

freshwater and estuarine species. It prefers to live in shallow, still waters with sufficient

vegetation. Optimal growth is achieved at 28-36°C, with lower and upper lethal temperatures

of 11-12°C and 42°C, respectively. It is an omnivorous grazer, naturally feeding on

phytoplankton, periphyton, aquatic plants, small invertebrates, benthic fauna, detritus and

bacterial films associated with detritus. They either feed via suspension filtering or surface

grazing. They also exhibited trophic plasticity according to the environment and the other

species they coexist with, (FAO, 2014; Fishbase Organization, 2012; Boyd 2014).

2.3 Fish Immune System

2.3.1 Fish Immune System, Innate Defenses and AMPs in General

The immune system protects organisms from diseases by identifying and eliminating

pathogens and suppressing the emergence of tumors. In addition, the immune system

participates in maintaining homeostasis during growth and development, and after

inflammatory responses or occurrences of tissue damage, (Magnadottir, 2010).

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Fish immune system is divided into the innate and adaptive systems, (Uribe, Folch,

Enriquez, & Moran, 2011). The innate immune response is divided into three compartments:

the epithelial and mucosal barrier, the humoral parameters and the cellular factors. Innate

parameters in general, rely to several pattern recognition proteins/receptors (PRP/R) to

recognize pathogen associated molecular patterns (PAMPs). Important PRRs include the toll-

like receptors (TLRs). PAMPS are associated with pathogens as well as inherent danger

signals from malignant tissues or apoptotic cells. PAMPs include various polysaccharides and

glycoproteins like bacterial lipopolysaccharide (LPS), flagellins, teichoic acid and

peptidoglycan, DNA CpG motifs and virus associated double stranded RNA (dsRNA),

(Magnadottir, 2010). Various internal and external factors highly influence innate responses.

Changes in temperature, exposure to stress in management and density have shown to have

suppressive effects on innate responses, while food additives and immunostimulants enhance

their efficiency, (Uribe, Folch, Enriquez, & Moran, 2011).

In contrast, acquired responses recognition depends on antigen receptors, which

possess random but narrow specificities, (Pellitero, 2008). However, adaptive responses in

fishes are limited due to their poikilothermic nature, limited repertoire of antibodies and slow

proliferation, maturation and memory of their lymphocytes, (Uribe, Folch, Enriquez, &

Moran, 2011).

Fishes are constantly exposed to pathogen-filled environments (Uribe, Folch,

Enriquez, & Moran, 2011) hence, they highly depend on their innate immune system for

survival, especially, their epithelial and mucosal skin barrier. This compartment provides

physical and mechanical protection to the fish. It also contains several immune defense

parameters including antimicrobial peptides (AMPs), complement factors and

immunoglobulin, (Magnadottir, 2010).

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The key factors to fish innate defense are the antimicrobial peptides (AMPs). They are

low molecular weight cationic polypeptides that have the ability to break down bacterial cell

walls, (Uribe, Folch, Enriquez, & Moran, 2011). They have the affinity to destroy bacterial

membranes by forming pores with barrel-stave, carpet, or toroidal pore mechanisms. AMPs

primarily rely on electrostatic interactions and biochemical structures. (Peng, Lee, Hour, Pan,

Lee, & Chen, 2012). AMPs block lytic bacterial enzymes and growth inhibitors such as

transferrin and interferon, (Magnadottir, 2010). AMPs in teleosts have been found primarily

in the mucus, liver and gill tissues, (Uribe, Folch, Enriquez, & Moran, 2011).

2.3.2 O. Niloticus Immune-Related Genes

Occurrence of diseases in an ecosystem limits any population, partially or completely,

depending on the degree of outbreak and the environmental conditions, (Real, 1996). In an

event of an attack by a parasite, an organism’s system naturally has its own defenses, which

ultimately involves immune-related genes. In this study, three Antimicrobial peptides were

selected, namely, Hepcidin, Piscidin-2 and Piscidin-3, due to their immune-related functions

described below.

2.3.2.1 Hepcidin

Hepcidin is a small cysteine-rich hormone secreted by the liver which is required for

iron level homeostasis in humans and other vertebrates. It is also called the Liver-Expressed

Antimicrobial Peptide (LEAP-1), as it may also act as an antimicrobial peptide. However the

in vivo activity of the peptide is unclear (Robertson, 2011 and Hsieh, Pan, & Chen, 2010). In

many fish species, the peptides’ gene structure and sequence is highly conserved. In addition,

evidence of antimicrobial and iron-regulatory functions has been observed, (Robertson,

2011).
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Hepcidins have been identified in the orders Perciformes, Cypriniformes,

Siluriformes, Gadiformes, and Salmoniformes; of which saltwater, freshwater, diadromous

fish representatives and fishes from extreme habitats are present, (Robertson, 2011).

Fish hepcidin is predominantly expressed in the liver. However, hepcidin expression

can also be detected in the spleen, kidney, blood, esophagus, stomach, intestine, heart,

muscle, brain, gonad, gill, and skin. The variety of the location where fish hepcidin is

expressed has led to the conclusion that it has antimicrobial functions other than iron-

regulatory functions, (Robertson, 2011).

Fish Hepcidin gene has three exons separated by two introns, which encodes a

prepropeptide consisting of a highly conserved signal peptide sequence (of 24 residues), an

acidic propiece (38-40 residues) and a mature peptide (19-27 residues), (Douglas et al. 2003

in Robertson, 2011). Fish Hepcidin proteins are divided into main clusters: the first group

possesses an amino-terminal sequence similar to the Q-S-H-L-S-L sequence in largemouth

bass (Micropterus salmoides) hepcidin-1 gene; while the other group lack this amino-

terminal sequence, (Robertson et al. 2009 in Robertson, 2011). It is hence possible that the

iron-regulatory hormone and antimicrobial functions are performed by separate proteins in

fishes, (Ganz 2006, Robertson et al. 2009 in Robertson, 2011).

Many fish encode multiple Hepcidin genes, including the close relative of O.

niloticus, Oreochromis mossambicus. O. mossambicus produces both Hepcidin(s) with a

sequence similar to Q-S-H-LS-L and Hepcidin(s) that lack this sequence (Huang et al. 2007,

Martin-Antonio et al. 2009, Robertson et al. 2009 in Robertson, 2011). Although information

that specifically describes the Hepcidin gene of O. niloticus is not yet established, it is

assumed that Hepcidin gene for both species are closely related.
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Expression of fish hepcidin is induced by exposure to Gram-positive bacteria, Gram-

negative bacteria, bacterial components such as lipopolysaccharides (LPS), infection,

inflammation, vaccination with attenuated bacteria, and polyI:C, a double-stranded RNA

(viral mimic), (Robertson, 2011). TH1-5 and TH2-3 are two of the hepcidins identified in

tilapia which display antimicrobial activity in vitro when stimulated by LPS. In contrast,

TH2-2, another tilapia hepcidin, is not stimulated by LPS and does not display antimicrobial

activity in vitro. TH2-3 is the tilapia hepcidin with an amino-terminal Q-S-H-L-S-L

sequence, while TH1-5 and TH2-2 are otherwise. TH1-5 is induced with Gram-positive

bacteria, while, TH2-3 is active against Gram-negative bacteria, (Huang et al. 2007 in

Robertson, 2011).

Infection of Aeromonas salmonicida to an Atlantic salmon induced an up-regulation

of hepcidin in the esophagus, stomach, pyloric caeca, liver, spleen, intestine, posterior

kidney, rectum, muscle, brain and heart, (Douglas et al. 2003 in Robertson, 2011). Gram-

positive bacterium Streptococcus iniae and Gram-negative bacterium Aeromonas

salmonicida induced Hepcidin expression in hybrid striped bass and white bass (Morone

chrysops), (Shike et al. 2002, Lauth at. al. 2005 in Robertson, 2011).

2.3.2.1 Piscidins: Piscidin-2 and Piscidin-3

Piscidins are cationic antimicrobial peptides, primarily expressed by mast cells. The

piscidin family is composed of structurally related mature peptides of 24~44 residues, having

an amphipathic -helical structure, which may correlate to the family's bactericidal activities

against a variety of microorganisms, (Peng, Lee, Hour, Pan, Lee, & Chen, 2012). Structure of

five piscidins (TP1~5) from Nile tilapia is shown in Figure 2.2.

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Figure 2.2 Three dimentional -helical structures of five different Nile tilapia piscidins (Peng,
Lee, Hour, Pan, Lee, & Chen, 2012).

Currently, about eleven species from the fish families, namely, Moronidae,

Sciaenidae, Sparidae, Latidae, Siganidae, Belontidae, Cichlidae, and Perichthyidae have been

identified to have AMPs from the piscidin family, (Peng, Lee, Hour, Pan, Lee, & Chen,

2012).

Described below are the Nile tilapia piscidins of interest, Piscidin-2 and Piscidin-3,

which was identified and differentiated by Peng et al. 2012. Piscidin-2 (TP2) and Piscidin-3

(TP3) cDNAs encode 77 and 76 amino acid residues, respectively. Both piscidins are 22

amino acids long and have a highly conserved, histidine-rich, phenylalanine-rich N-terminus

and a variable C-terminus (Figure 2.3). TP2 possesses a four exon-three intron structure,

similar with TP1, -4 and -5, while Tp3 gene possesses a three exon-two intron structure.
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Figure 2.3 Amphipathic -helical structures of Tilapia Piscidin 2 (TP2) and Piscidin 3 (TP3)

High levels of an AMP in a tissue correlate to immune function of that AMP in that

area. Comparative quantitative RT-PCR analysis to study mRNA expression levels in

different tissues showed that TP2 mRNA was most abundant in the skin, head, kidneys and

spleen and lowest in the intestines, while TP3 mRNA was most abundant in the skin, head,

kidneys and gills, (Peng, Lee, Hour, Pan, Lee, & Chen, 2012).

Levels of Nile tilapia piscidin mRNA expressions in different tissues after being

stimulated with V. vulnificus and S. agalactiae after 24 and 48 hours post-infection was also

quantified by Peng et al. 2012. Initial downregulation of TP2 levels (24 hours) was recorded,

then, upregulation was observed after 48 hours. Conversely, initial upregulation and late

downregulation was observed for TP3.

The antimicrobial spectrum of synthetic tilapia piscidins-2 and -3 against selected

gram-positive and gram negative bacteria were also determined by Peng et al. 2012. It was

found that TP3 presented excellent antimicrobial activity towards V. vulnificus, A.

hydrophila, V. alginolyticus, P. aeruginosa, S. agalactiae, E. faecalis and S. agalactiae, in

contrast to TP2, which exhibited no antimicrobial activities. The difference in antimicrobial

activities was assumed to be correlated with amphipathicity and cationicity. Hemolytic

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activities in tilapia red blood cells were also assayed. TP3 were 42% hemolytic in fish RBC

at 100 g/ml, while TP2 achieved 60% hemolysis at 40 g/ml.

Literature that describes antibacterial activity of TP2 is limited. Yet, evidences of

antifungal and antiparasitic activities are established. Zahran and Noga, 2010 provided an

evidence of Piscidin-2 antifungal activity against water mold Saprolegnia, an important

pathogen for freshwater fish. Antiparasitic effects of Piscidin-2 were also observed against

three protist ectoparasites of marine fish: the ciliates Cryptocaryon irritans and Trichodina

sp., and the dinoflagellate Amyloodinium ocellatum and one ciliate ectoparasite of freshwater

fish: Ichthyophthirius multifiliis, in the study of Colorni et al. 2008. Information on the

antibacterial activity of TP2 or Piscidin 2 in general, may be provided in this study.

2.4 Pathogens: Aeromonads (A. hydrophila, A. caviae, A. veronii biotype sobria)

Similar with all other organisms, O. niloticus are often challenged by pathogens.

Several species of Aeromonas are frequently associated to major die-offs and fish kills

around the globe, including major and minor aquacultures in the Philippines, resulting to

enormous economic losses, (Janda & Abbott, 2010) and (Yambot, 1998). Members of genus

Aeromonas are Gram-negative, non-spore-forming, rod-shaped, facultatively anaerobic

bacteria. They are differentiated from the members of Enterobacteriaceae for being oxidase-

positive. When incubated at 25°C, Aeromonas strains usually produce tan to buff-colored

colonies on Trypticase soy agar (Becton-Dickinson, Cockeysville, Md.), (Abbott, Cheung, &

Janda, 2003).

The Aeromonads occur ubiquitously and autochthonously in aquatic environments,

(Sartori, 2009). They are isolated from rivers, lakes, ponds, seawater (estuaries), drinking

water, groundwater, wastewater, and in sewage of various treatment stages, (Janda & Abbott,

2010). Moreover, they can be isolated from every environmental niche where bacterial
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ecosystems are present, including food, domesticated pets, invertebrate species, birds, fishes,

ticks and insects, natural soils and etc., (Janda & Abbott, 2010). The presence of organic

matter in these sources facilitates an increase in number of these organisms, (Malupig, 2004).

Studies on the natural habitats of the Aeromonads by Hazen, et al. in the 1980s led to

the assessment that Aeromonas prefer lotic than lentic systems, which is usually the case for

aquaculture systems. In addition, the organisms were found to be at optimum in thermal

gradients ranging from 25°C to 35°C. They are capable of growing over a wide range of

temperatures, conductivities, pHs, and turbidities, and is only limited in extreme habitats,

such as, extremely saline environments, thermal springs, and highly polluted waters, (Janda

& Abbott, 2010).

The role of Aeromonas as the causative agents of fish diseases has been determined

for decades, prior to the identification of their role in causing systematic diseases to humans.

Although they are normal flora in the fishes’ intestines, Aeromonas become pathogenic either

as sole or co-pathogens. They cause invasive secondary infections in immunosuppressed fish,

due to spawning or undesirable environmental factors. In addition, these pathogens also host

on amphibians, reptiles, mollusks and arthropods, (Gosling, 1996) and (Janda & Abbott,

2010). Previous publications have indicated that the Aeromonas genomospecies (A.

hydrophila, A. caviae, and A. veronii bv. sobria) are responsible for the vast majority (85%)

of human infections and clinical isolations attributed to this genus. In freshwater ecosystems

specifically, A. hydrophila is one of the predominant species present, A. caviae is common,

while A. veronii have had rare reports (Janda & Abbott, 1998 in Janda & Abbott, 2010).

A. hydrophila have been reported to cause Motile Aeromonas Septicemia (MAS),

ascitis, erosion, ulceration, detachment of scale and exophthalmia to O. niloticus. Moreover,

the bacteria also cause varying degrees of other systemic infections to O. niloticus including
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congestion and focal lesions in the liver, spleen, and kidney, (Ibrahem, Mostafa, Arab, &

Rezk, 2008). In the Philippines, disease symptoms associated with A. hydrophila that were

observed also included skin lesion, fin rot, discoloration, mouth sore, eye opacity, dislodged

eyeball and sluggishness, (Yambot, 1998). Post-mortem analysis of septicemic and ulcerated

O. niloticus individuals was also found to be caused by other members of the genus

Aeromonas namely, A. caviae and A. veronii biotype sobria, (Shayo, Mwita, & Hosea, 2012).

Implications of disease vary, which may range from sudden death in otherwise healthy fish,

to lack of appetite, swimming abnormalities, group exclusions, pale gills, bloated appearance

and skin ulcerations, (Swann and White, 1989 in Claus, 2013). Nevertheless, the incubation

and severity of disease is influenced by several factors including the pathogens' virulence, the

kind and degree of stress exerted on a population by its environment, and the physiologic

conditions of the host and their inherent genetic resistance, (Cipriano, 2001 in Claus, 2013).

Specifically, the incubation period varies from 2 to 4 days in natural setting and 8-48 hours in

experimental infection models, (Yardimci and Aydin, 2011 in Claus, 2013).

The Aeromonas genus pathogenicity is multifactorial, which includes exoenzymes

(amylase, DNAse, RNAse, esterase, lipase, gelatinase, protease, chitinase, mucinase and

hyaluronidase, among others), enterotoxins, cytotoxins, cytotonic toxins, haemolysins,

adherence and invasiveness, (Malupig, 2004). A. hydrophila's resistance to hosts' non-specific

resistance is attributed to the production of several toxins including haemolysins and

enterotoxin, possession of S layer, the ability to internalize, serum resistfance and the

resistance to phagocyte-mediated killing, (Zhang et al., 2000 in Claus, 2013). A. caviae

specifically possess many putative virulence genes, including those encoding a type 2

secretion system, RTX toxin, and polar flagella, (Beatson, et al., 2011). Virulence factors of

A. veronii include aerolysin, lipase, cholesterol acyltransferase, serine protease, structural

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gene flagellin and class I integrons, that contribute resistance to multiple antibiotics, (Nawaz,

et al., 2009).

In reality, most intensive tilapia production systems are caused by multiple disease-

causing agents. Studies on tilapia diseases to date are usually focused on a single pathogen.

The possibility of a single pathogen resulting in death-loss may be minimal. (Shoemaker, Xu,

Klesius, & Evans, 2008). Concurrent infections have been proven to cause increased

mortality. Shoemaker, et al. 2008 infected Nile tilapia with Gyrodactylus niloticus and S.

iniae. Mortality was significantly higher with the concurrent infection (42%), as compared to

immersion infection with S. iniae alone (7%) and parasitism with G. niloticus only (0%).

Consequently, vaccinating Nile tilapia with a combination of pathogens should further

stimulate protective immunity. Lin, et al. 2005, for instance, vaccinated Cobia (Rachycentron

canadum), a warm water fish, with three inactivated pathogens: Vibrio alginolyticus, Vibrio

parahaemolyticus and Photobacterium damselae subsp. piscicida. Survival rate after one

vaccination was 86-92%. In this study, the previously mentioned Aeromonads shall then be

inactivated and combined into one vaccine.

2.5 Immunization and Vaccination

Vaccination is the method by which an organism's immune system is stimulated to

develop immunity against a pathogen, by administration of antigenic material. The main

purpose of vaccination is to prevent diseases. The associated economic objective of

vaccination is to ensure that, on average, the cost of the vaccines purchased, their application,

and any loss of productivity caused by their application, is less than the cost of the disease if
 19

vaccines are not used, (Responsible Use of Medicines in Agriculture Alliance (RUMA),

2006).

In actuality, it is difficult to prevent fishes from contacting their environmental

pathogens, or pathogens of wild fish origin when they are kept in management methods such

as sea cages or ponds and raceways. Unlike other types of livestock, there are limited

opportunities in which vaccines can be administered in the lifetime of a farmed fish. The

earliest point in a fish’s where it can be vaccinated is when it becomes immunocompetent

(some time subsequent to when the yolk sac has been absorbed). However, administration

during this period is extremely tricky. Vaccine administration is hence limited by the physical

size of the fry and may only be possible by the immersion or oral routes which are limited in

their duration of immunity. In addition the minimum sizes for fish injection vaccination is

usually determined by the companies developing the vaccines and the size of fish in which

they were developed, (Responsible Use of Medicines in Agriculture Alliance (RUMA),

2006).

There are four types of vaccines usually used in farmed fishes: the inactivated

bacterial vaccines, inactivated viral vaccines, subunit (derived from recombinant technology)

vaccines and DNA vaccines. Since farmed fish are not usually kept in isolation from their

wild counterparts, the use of live modified or attenuated products is not encouraged,

(Responsible Use of Medicines in Agriculture Alliance (RUMA), 2006).

Since the pathogens of interest are of bacterial nature, this study shall utilize

inactivated bacterial vaccines. Inactivated bacterial vaccines are produced by fermenting

pathogens in large quantities and then introducing inactivating agents such as formalin.

Although killed, these organisms still retain the original antigenic characteristics of the live

bacteria since their basic shape and structure has not been altered. Only their ability to grow,
 20

reproduce and cause infection to the host is terminated, (Responsible Use of Medicines in

Agriculture Alliance (RUMA), 2006).

Quality control test for an inactivated vaccine is especially important to confirm that

the bacteria have indeed been killed. The major disadvantage of inactivated bacterial vaccines

is that it does not produce a long-term immunological response, and hence will not induce

long-term protection to the disease. A remedy for this is to combine a compound, specifically

an adjuvant, which improves the presentation of the antigen to the immune cells of the fish

and further encourages the antigens to persist within the body cavity of the fish to improve

the duration of protection, (Responsible Use of Medicines in Agriculture Alliance (RUMA),

2006).

In this study, fingerlings and grow-outs were selected. Vaccination of fingerlings shall

be done by immersion, while the grow-outs are to be vaccinated by injection, (Iwama &

Nakanishi, 1997). Injection method has been proven to be effective in providing best

protection, however, it is not practical to routinely immunize smaller fishes by injection,

(Iwama & Nakanishi, 1997), (Responsible Use of Medicines in Agriculture Alliance

(RUMA), 2006).

2.6 Gene-expression, Quantification and Real-Time Polymerase Chain Reaction

The genetic material of any cellular organism is the DNA. Information that is encoded

in the DNA is utilized in the synthesis of a functional gene product. Usually, these products

are proteins, which are made up of strings of amino acids joined together by peptide bonds.

The type of proteins that a cell synthesizes determines the type and functionality of the cell.

Hence, gene expression is the most fundamental level in which genotype gives rise to
 21

phenotype, (Tamarin, 2001). Assay on gene expression has its advantage, compared to that of

protein assay. The former can be performed by directly obtaining tissues or cells from

animals. In contrast, the latter requires in vitro cell culture, in some cases with mitogens,

often resulting in the production of cytokines not originally produced by the cells in vivo,

(Gause & Adamovicz, 1994).

Scientific advancement has led to the discovery of several techniques that allow to

measure or quantify the changes in gene expression. These techniques include the Northern

blot, the RNase protection assay, in-situ hybridization, and the Real-Time Polymerase Chain

Reaction (RT-PCR or qPCR). The Northern blot technique or the more sensitive RNase

protection assay has been sufficient in quantifying gene-expression between samples.

However, in instances where the quantity of the sample is low, the said techniques are no

longer practical. Instead, the more sensitive RT-PCR is employed, (Gause & Adamovicz,

1994).

In RT-PCR, an RNA sample is initially reverse-transcribed into a cDNA, with the

desired target amplified using specific primers. Successful amplification can performed with

only less than 10 copies of target RNA. The sensitivity of the technique is an attribution of

the chain reaction, in which the products from one cycle of amplification becomes the

substrate for the next, enabling exponential increase of product, (Gause & Adamovicz, 1994).

The major difference between traditional PCR and RT-PCR is that, the amount of

PCR product in the former is only measured at the end point of amplification, while the PCR

products in the latter, are measured preceding each round of cycle. Amplification of these

products are measured as they are produced using a fluorescent label. The fluorescent dye

molecules binds, either directly or indirectly via labeled hybridizing probe, to the

accumulating DNA molecules. These fluorescence values are recorded during each cycle of
 22

amplification. The linear correlation between the PCR product and fluorescence intensity is

used to calculate the amount of template present at the beginning of the reaction, (Sigma-

Aldrich, 2014).

There are two popular methods used in analyzing data from RT-PCR experiments,

namely, the absolute and relative quantification. Absolute quantification determines the exact

transcript copy number, usually by relating PCR signals to a standard curve. The main

disadvantage of this method is the difficulty in the generation of standard curves. Conversely,

relative quantification relates PCR signal of target transcript to that of another sample such as

an untreated control or a calibrator or internal control gene. In measuring the relative changes

in gene expression, certain equations, assumptions and tests are needed to properly analyze

the data. The comparative CT method or the 2-∆∆CT method is usually utilized in analyzing relative

gene expression, (Livak & Schmittgen, 2001). CT value or threshold value is the point at which

fluorescence is first detected as statistically significant above an arbitrarily placed threshold. The CT

value is inversely correlated to the logarithm of the initial copy number. It is imperative that the

threshold is set above the amplification baseline and within the exponential increase phase.

Theoretically, there is an equal number of molecules present in all of the reactions at any given

fluorescence level. Consequently, it is hence assumed that at the threshold level, all reactions contain

an equal number of specific amplicons, (Sigma-Aldrich, 2014).

The mRNA levels are generally normalized, using standard or calibrator genes, to

minimize room for deviations in the process of measurement which may come from the

variations of executions, disparities in mRNA quality and quantity between samples due to

pipetting errors, or variations in the efficiency of enzymes, (Banda et al. 2007 in Rebouças,

Costa, Passos, Passos, van den Hurk, & Silva, 2013). Reference genes or housekeeping genes

are expressed in variety of cells and tissues that do not exhibit changes in expression under

experimental conditions or disease-state. There is no ideal housekeeping gene, as they show

 23

variability in expression levels in different tissues (Vandesompele et al. 2002 in Rebouças,

Costa, Passos, Passos, van den Hurk, & Silva, 2013). Yang, et al., 2013 evaluated the

common reference genes for RT-PCR in Nile tilapia, and results exhibited this tissue-

correlated variation.

The most widely used gene for gene-expression normalization is β-actin, a gene that

encodes for a structural protein cytoskeleton, (Rebouças et al. 2013; Ma, et al. 2014 and Yan,

et al. 2012). The gene is primarily expressed in Nile tilapia thymus (Nithikulworawong,

Yakupitiyage, Rakshit, & Srisapoome, 2012), however, expression is also observed in the

liver, spleen, kidney, brain, heart, muscle and intestine, (Yang, et al., 2013).
 24

III. METHODOLOGY

The methodology outlines the procurement and preparation of Nile tilapia and

Aeromonad pathogens, vaccine preparation, vaccination by immersion and injection, tissue

collection, infection with Aeromonas, RNA analysis, cDNA extraction, qPCR and analysis,

and the statistical treatment. Below is an experimental design that outlines the

experimentation and analysis process.

3.1 Nile tilapia and Aquaria Set-up

3.1.1 Procurement of Nile Tilapia fingerlings

Approximately 500 size-24 GIFT (Genetically

Improved Farmed Tilapia) fingerlings and eighty 20-60

grams tilapia grow-outs are to be procured from Bureau of

Fisheries and Aquatic Resources, Central Luzon State

University (BFAR-CLSU), Science City of Muñoz, Nueva Ecija.

3.1.2 Acclimatization Period

The fishes -- fingerlings and grow-outs separated, are to be transferred in an aquarium

of size 35.5 x 17.5 x 17.5 inches for the acclimatization period. They are initially placed in

one tank during acclimatization to remove the bias and variation of environmental

parameters. This acclimatization period of three weeks is a period for the fishes to grow and
 25

will especially ensure that the fingerlings are not limited by stress due to change of habitat or

microbial infections. This will further ensure that the deaths during or after the immunization,

if present, are not caused by the said stresses. The glass tank is to be supplied with 100 L

dechlorinated water, with a constant 12:12 hour light: dark period and supplemented with

three air stone aerations. The fingerlings are to be fed with commercial diet amounting 10%

of their total body weight, four to six times a day, while the adults are to be fed three times a

day, amounting about 3% of their total body weight. Prior to immunization, abnormalities on

the behavioral and physical characteristics of the fishes are to be noted, if present. TRS®

vacuum salt at 5ppt is to be placed in the tank to prevent fungal growth and to alleviate fish

from osmotic problems, especially after having been transported and transferred, (Lass,

2013).

3.1.3 Experimental Set-up

120 Nile tilapia fingerlings and 60 grow-outs are to be randomly picked from the

acclimatization tanks using fish scoop. Three tanks (size: 35.5 x 17.5 x 17.5 inches) each are

allotted for the control group and the treated/immunized group, representing three replicates.

Twenty (20) fingerlings are to be placed in each fingerling tank, while ten (10) fishes are

placed in each grow-out tank. Physico-chemical parameters of the tanks are to be taken

and/or checked daily, ideally without deviation: similar amount of dechlorinated water,

maintenance of 12:12 hour light: dark period, temperature, pH and salinity, and similar

source and amount of aeration. They shall have a commercial diet amounting 10% of

fingerling total body weight, and 3% of grow-out total body weight.

3.2 Bacterial Culture and Preparation

Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii biotype sobria are

to be obtained from isolations obtained from reported fish-kills in Sto. Domingo, Mexico,
 26

Pampanga, BFAR-CLSU and Mañanggo, Pampanga, respectively. They are to be maintained

and cultured in the Molecular Biology and Biotechnology Laboratory of the College of

Fisheries in CLSU. Each of the Aeromonads shall be cultured using Trypticasein Soy Agar

(TSA) (40g/L). They shall be incubated for five days since the Aeromonads generally grow

slow.

After incubation the bacteria are to be harvested from the plates using inoculating

loops. They are to be suspended with 1.5mL PBS (Phosphate Buffer Saline Solution) and

agitated until the bacterial solutions become homogenous. The respective concentrations of

the pathogens are to be measured at A260nm/A280nm using NanoDrop 2000c

SpectophotometerThermoscientific, USA.

The desired concentration of pathogens for vaccine formulation is at 107 CFUs/ml.

McFarland’s Standard shall serve as a basis to attain this concentration. In McFarland’s

standard, 108 CFUs/ml has a A260nm/A280nm value of 0.6. To attain 107 CFUs/ml, serial

dilution shall be employed. PBS solution shall be utilized to dilute the solution. If the desired

concentration is attained (107), preparation of vaccine shall proceed.

3.3 Vaccine Preparation

The Aeromonads shall be inactivated using formalin, amounting 4% of the total

pathogen-PBS solution. The solution shall then be centrifuged at 13K rpm, to isolate

pathogen associated molecular patterns (PAMPs). The solution shall be cleansed of formalin

by washing it with PBS solution three times. After thorough mixing, the solution shall be

inoculated in TSA-plates to check if the pathogens are still active. If no bacterial growth is

observed after five days, the vaccination shall proceed. The solutions shall then be combined

in one sterile container in preparation for the vaccination.

 27

3.4 Vaccination

Prior to the vaccination of a fish set, they are placed in a sterile basin with MS-222

(Tricaine Methane Sulfonate) to sedate the fishes.

3.4.1 Immersion

The fingerlings shall be vaccinated by immersion. About 1 Liter of the Aeromonas

vaccine shall be utilized for the immersion of each set-up. The fingerlings shall be immersed

in the vaccine for 20 minutes and shall be returned to their respective tanks after the

immersion.

3.4.2 Injection

The tilapia grow-outs shall be vaccinated by injection. About 0.1 mL of the vaccine

shall be injected intraperitoneally into the fishes. They shall then be immediately returned to

their respective tanks.

3.5 Tissue Collection

Fishes are to be collected prior to the immunization, two weeks (14th day),

three weeks (21st day) and four weeks (28th day) after immersion and injection. In every

collection, five fishes are to be taken from each fingerling tank, while two fishes are to be

taken from each grow-out tank. Tissues shall be obtained from these fishes, where RNA shall

be extracted. Each fish is to be randomly caught using a scoop net and placed in a basin

containing MS222 (Tricaine Methane Sulphate/300mg per L). Kidney tissue of the fishes is

to be collected since it is an organ where Hepcidin, Piscidin-2 and Piscidin-3 are all primarily

expressed. For temporary storage, the samples are to be placed in ultra-low temperature

freezer (-80°C) or liquid nitrogen, to prevent RNAse enzymes from degrading RNA.
 28

3.5.1 Tissue collection for Fingerlings

Using iris scissor, the fingerlings’ heads and tails are to be excised. The ventral

portion of the fish, including a portion of its trunk and its innards are to be removed, leaving

only with the fish kidney area and the muscles surrounding it. These tissues are to be placed

in 1.5mL microcentrifuge tube.

3.5.2 Tissue collection for Tilapia grow-outs

Using iris scissors, kidney tissue is to be isolated by opening the fish abdomen and

excising the kidney (which should appear as dark linear organs that run the entire length of

the peritoneal cavity). The tissue is to be pulled using forceps. Approximately 30mg kidney

sample was collected and placed in a 1.5 ml microcentrifuge tube labelled and stored.

3.6 RNA Extraction

Tissue samples of the control and experimental group are to be subjected to RNA

extraction using the RNA extraction kit SV Total RNA Isolation System, Promega, USA.

Detailed protocol can be found in the Appendix. The concentration and quality o f the RNA

obtained is to be measured using NanoDrop 2000c SpectophotometerThermoscientific, USA,

at A260nm/A280nm.

3.7 cDNA Synthesis

RNA is to be reversed-transcribed using iScriptcDNA Synthesis kit (Bio-RAD, CA,

USA), as stated in the manufacturer’s instruction. Reverse transcription reaction mix is to be

prepared by combining the following components listed in Table 3.1.

 29

Table 3.1.Components of Reverse Transcription Reaction Mix

Components Volume per Reaction
5x iScript reaction mix 4 µl
iScript reverse transcriptase 1 µl
Nuclease-free water Variable
RNA template Variable
Total Volume 20 µl
The components shall be mixed in a new sterile microcentrifuge, following order

specified above. PCR reaction shall be performed using a PCR thermal cycler with thermal

profile of 5 minutes at 25 oC, 30 min at 42 oC, 5 min at 85 oC then hold at 4 oC.

3.8 qPCR and Analysis

Gene specific primers for Hepcidin, Piscidin-2 and Piscidin-3 in Nile Tilapia and β-

actin gene (shown in Table 3.2) were used. The β-actin is to be utilized as the reference or

calibrator gene in the RT-qPCR analysis.

The quantitative assays for Hepcidin, Piscidin-2 and Piscidin-3 are to be done using

LightCycler 480 (Roche Applied Science, Germany). The thermal conditions for the genes

are ran in 96-well plates under thermal condition of 95°C for 5 mins at followed by 40 cycles

of 15 sec at 96°C, 1 min at 60°C, 45 sec at 72°C, and 2 min at 72°C and melting curve of 5

sec at 95°C, 1 min at 65°C, continuous at 98°C, and cooling 10 sec at 40°C.

Three replicates for each sample are to be run. Each replicate, amounting a total of 20

µl, shall contain 3 µl nuclease-free water, 2 µl primer (forward and reverse) (Sequence in

Table 3.2), 10 µl master mix and 5 µl DNA (cDNA) template. RT-PCR products of target

genes and control gene are to be ran in gel electrophoresis and are to be sequenced to confirm

their identity.

β-actin shall be used for normalization or as a housekeeping gene. It shall also be

quantified along with the genes of interest. The Livak-mothod, described below, shall be

utilized to analyze the data from the assay.

 30

Table 3.2. Summary of Gene Specific Primer for Each Genes

Gene Forward Primer Reverse Primer
Hepcidin 5’- ATG AAG ACG TTC AGT GTT GCA GTT 5’- TCA GAA CCT GCA GCA AAC -3’
GCA GTT GCA GTG -3’
Piscidin-2 5’- AGC CAA ACA TTT TCT TCA TCG -3’ 5’- AAC GAA AAC AAC CTG TAG GAA -3’

Piscidin-3 5’- GGG AGG CCT TTA TTC ACC AT -3’ 5’- TGC TGC TGT TGT TGC TGT TT -3’

β-actin 5'- CGG AAT CCA CGA AAC CAC CTA -3’ 5'- TTG CTG ATC CAC ATC TGC TGG -3’

3.8.1 Analysis of Data Using Livak or 2-∆∆CT Method

Livak's method is commonly utilized to determine the difference in expression level

of target genes in different samples. The CT of the target gene is to be normalized to that of a

reference/housekeeping gene using Equation 3.1. The ∆ACT of the sample shall then be

normalized to the ∆ACT of the calibrator using the ratio in Equation 3.1. The expression ratio

2-∆∆CT or Normalized Expression ratio shall then be calculated.

Equation 3.1 (1) ∆CT (test) = CT (target, test) – CT (ref, test)

(2) ∆∆CT = ∆CT (calibrator)

(3) 2-∆∆CT = Normalized Expression ratio

3.9 Statistical Treatment

Statistical significance of gene-expression between immunized and unimmunized

individuals shall be tested using Student’s T-Test, at 95%. In addition, significance of the

expression by each gene in the control and treated set-ups shall be tested using One-Way

ANOVA. The statistical significance of expression from different time-periods (day of pre-

immunization and days of post-immunization) shall also be determined using One-Way

ANOVA. A P-value <0.05 shall be considered significantly different.

 31

IV. EXPECTED RESULTS

The innate responses of fishes, some of which brought about by Hepcidin, Piscidin-2

and Pscidin-3, are vital factors in combating pathogens, and are much more efficient and less

limited compared to their adaptive immune responses, (Uribe, Folch, Enriquez, & Moran,

2011). It is predicted that Hepcidin, Piscidin-2 and Piscidin-3 expression will up-regulate

after infection (Colorni, Ullal, Heinisch, & Noga, 2008); (Robertson, 2011). Gene-expression

of Aeromonad-imunized individuals is also expected to be higher than that of unimmunized

individuals.

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