Research papers- Chapter 1 to 5Document Transcript
1 CHAPTER I INTRODUCTION This chapter presents the background of the problem,main problem, the sub-problems, and hypothesis,significance of the study and the scope and limitations ofthe study.Background of the Problem Nile Tilapia (Oreochromis niloticus) is a very popularaquaculture species in the Philippines at present andconsidered as an “aquatic chicken” offering economical andsocial benefits mainly for rural communities. It also playvital role in terms of worldwide employment, however therewere reported cases of high mortality rates in differentspecies of Tilapia cause by Aeromonas species (Badillo,2010). One of the most common bacteria that infect the wildand cultured Tilapia is Aeromonas sobria. Aeromonas sobriais water borne pathogen that are common in almost allaquatic environments including fresh, brackish and marinewater. They cause fin rot or skin rot disease and may leadto heavy mortality in cultured tilapia (El-Sayed, 2006). Aeromonas sobria veronii also causes a similar diseasein fish including Motile Aeromonas Septicemia in Tilapia(Janda and Abbott, 2010).
2 Bacterial infections, caused by motile members of thegenus Aeromonas are among the most common and troublesomediseases of fish raised in ponds and recirculating systems. The Motile Aeromonas infections have been recognizedfor many years and have been referred to by various names,including Motile Aeromonad Septicemia (MAS), MotileAeromonad Infection (MAI), hemorrhagic septicemia, redpest, and red sore (Camus, Durborow, Hemstreet, Thune andHawke, 2012). One of the most common treatment for the skin lesions,fin rots and infections caused by Aeromonas sobria is theuse of chemicals specifically tetracycline. However, in2012, Romero, Feijoo and Navarette stated that fish do noteffectively metabolize antibiotics and will pass themlargely unused back into the environment in the form offeces. It also been estimated that 75% of antibiotic fed tofish are excreted into the water. In 2000, Cruz-Lacienda, Dela Pena, Lumanlan-Layo alsostated that chemical control such as antibiotics may leadto development of drug resistance bacterial strain throughoveruse and misuse of antimicrobial. Regarding to these problems, there is an urgentdiscovery of new drugs and alternative therapies to controlbacterial diseases.
3 According to the study of Krishnaiah et al. 2009 ascited by Namuli, Abdullah, Sieo, Zuhainis and Oskoueian2011, plant secondary metabolites (alkaloids, terpenoidsand phenolic compounds) are potential antimicrobial agentsthat can help to alleviate problem of antibioticresistance. Treatments of bacterial diseases with medicinal plantshaving antibacterial activity are potentially beneficialalternative in aquaculture (Pandey, Sharma and Mandloi,2012). Jatropha curcas (Tuba-tuba), belongs to the FamilyEuphorbiacaea, has been initially considered a traditionalherb in many parts of the world. Different parts ofJatropha curcas have been used in treating different formsof infection. According to Igbinosa et al. and Akinpelu et al.(2009) as cited by Namuli, Abdullah, Sieo, Zuhainis andOskoueian, 2011, the presence of Flavonoids and saponin inJatropha curcas stem bark and leaves extract respectivelywas observed also gallic acid has been reported in theleaves extract of Tuba-tuba plant. Tinospora crispa (Makabuhay), belongs to the FamilyMenispermaceae, is a climbing vine plant. Leaves yielded
4picroretine, traces of an alkaloid. Both are found to be anantibacterial agent. Hence, this study aimed to develop an alternativeantibacterial agent from Jatropha curcas (Tuba-tuba) andTinospora crispa (Makabuhay) against Aeromonas sobriaveronii causing Motile Aeromonad Septicemia in Oreochromisniloticus (Nile Tilapia) also at the same time it aimed notto harm both fish and environment.Statement of the Problem The study dealt with the determination ofantibacterial activity of leaf extract of Jatropha curcas(Tuba-tuba) and Tinospora crispa (Makabuhay) against thefish pathogen Aeromonas sobria veronii. Specifically, the study answered the following subproblems: 1. What is the mortality rate of Tilapia fingerlings in different concentrations of: a. Jatropha curcas(Tuba-tuba) (0.13% and 0.11%) b. Tinospora crispa (Makabuhay) (0.0045% and 0.0040%)
5 c. Negative control (0%) 2. What is the zone of inhibition of the following concentrations of: a. Jatropha curcas (Tuba-tuba) and Tetracycline (0.13%, 0.11%, 0.09%, 0.07% and 0.05%) b. Tinospora crispa (Makabuhay)and Tetracycline (0.0045%, 0.0040%, 0.0035%, 0.0030% and 0.0025%)? 3. Is there a significant difference among the zones of inhibition of: a. Jatropha curcas (Tuba-tuba) and Tetracycline (0.13%, 0.11%, 0.09%, 0.07% and 0.05%) b. Tinospora crispa (Makabuhay)and Tetracycline (0.0045%, 0.0040%, 0.0035%, 0.0030% and 0.0025%)?4. What is the best leaf extract that will inhibit thegrowth of Aeromonas sobria veronii between Jatropha curcas(Tuba-tuba) and Tinospora crispa (Makabuhay)?
6Hypotheses of the study 1. There is no significant difference among the zones of inhibition of: a. Jatropha curcas (Tuba-tuba) and Tetracycline (0.13%, 0.11%, 0.09%, 0.07% and 0.05%) b. Tinospora crispa (Makabuhay)and Tetracycline (0.0045%, 0.0040%, 0.0035%, 0.0030% and 0.0025%)?Significance of the Study Asia contributes more than 90% of the world’saquaculture production including China, Thailand,Indonesia, Malaysia, Lao Peoples Democratic Republic,Costa Rica, Ecuador, Colombia, Honduras, Brazil, Taiwan,and Philippines. In the Philippines, Aeromonas species were reported tobe the cause of high mortalities in reared Oreochromismossambicus, Oreochromis niloticus and Tilapia zillii, inthis case antibiotics specifically tetracycline, terramycinand other pharmaceutical drug have been used in treatment,however several side or harmful effects that affect bothfish and environment was observed (Fisheries and AquaticOrganization, 2011).
7 According to Shayo S.D, Mwita C.J and Hosea K., 2011,Infectious diseases of fish are responsible for significanteconomic losses in the wild and aquaculture venturesworldwide. This study helps to decrease the damages done toboth wild and farmed freshwater fish population leading tomortality and severe loss of income. The study aimed to develop an alternativeantibacterial agent from Jatropha curcas (Tuba-tuba) andTinospora crispa (Makabuhay) which do not harm both fishand environment. The study also emphasized the importance of plants intreating bacterial disease in fishes and will give anotherpotential usage of Jatropha curcas (Tuba-tuba) andTinospora crispa (Makabuhay) other than its usual usage. Harnessing the potentials of these plant materialsagainst fish bacterial pathogens will be a great help tothe aquaculture industry as a whole. The study will also inform the farmers about theharmful side effects of antibiotics in fishes specificallyTilapia and to the environment. Also, Farmers can have maximum usage of theiravailable resources such as those of Jatropha curcas andTinospora crispa instead of buying commercially preparedantibiotics, thus having least cost of expenses.
8 The study enhanced the researchers writing skills, aswell as their skills in microtechniques and will giveopportunity to the researchers to practice and developtheir laboratory skills.Scope and Limitation of the Study The study mainly focused on the antibacterial activityof Jatropha curcas (Tuba-tuba) and Tinospora crispa(Makabuhay) against Aeromonas sobria veronii causing MotileAeromonad Septicemia (MAS) in Oreochromis niloticus (NileTilapia). The study also involved the computation of the lethalconcentration (LC50) of leaf extract of Jatropha curcas(Tuba-tuba) and Tinospora crispa (Makabuhay). Moreover,Nile tilapia fingerlings were used as a subject fortoxicity test. The experiment used the leaves of Jatropha curcas(Tuba-tuba) and Tinospora crispa (Makabuhay) for aqueousextraction. The study was conducted at Philippine NormalUniversity and lasted for two months.
9Definition of Terms The terms are defined based on how they are used inthis study. The following terms are:Aquatic chicken- Tilapia is also called as Aquatic chickenbecause a range of systems is suitable for them.Antibiotic- This is the chemical control that prevent orinhibit the growth of bacteria.Antimicrobial agent- This are chemicals or other substancesthat either kill or slow the growth of the bacteria.Floating pellet- This is the food of the fish which iscommercially prepared.Lethal concentration- Is a concentration of a potentiallytoxic substance in an environmental medium that causesdeath following a certain period of exposure (denoted byLC).Lethal concentration 50 (LC 50) - It means that testsubstance concentration, calculated from experimentally-derived mortality data, that is lethal to 50 percent of atest population during continuous exposure over a specifiedperiod of time.Medicated Feed- It is a floating pellet with the presenceof antibiotics.Static Test Procedure- The test solution is not renewedduring the period of the test.
10Toxic- The degree of the substances that can harm Tilapia.Toxicity test- A test method designed to generate data thatcan harm the Tilapia.Zone of inhibition- This is an area around a paper disk orcolony of bacteria or mold where no other organisms aregrowing.
11 CHAPTER II REVIEW OF RELATED LITERATURE Presented in this chapter is a synthesis of facts thatsupports the following topics: Taxonomic Classification ofOreochromis niloticus (Nile Tilapia) ; Generalcharacteristics and occurrence of Aeromonas sobria veronii;Pathogenicity of Aeromonas sobria veronii in Tilapia;Chemical control of the bacteria in the field including themethods, advantages and disadvantages; Taxonomicclassification and Morphological description of Jathrophacurcas (Tuba-tuba) and Tinospora crispa (Makabuhay);Antimicrobial agents in plants; Chemical composition andToxicity of Jatropha curcas and Tinospora crispa . All ofwhich have significant bearing or relation to the problemunder investigation.Related LiteratureDescription and Taxonomic Classification of Oreochromisniloticus (Nile Tilapia)Kingdom: Animalia Plate 2.1: Oreochromis niloticusPhylum: Chordata (Nile Tilapia)Class: ActinopterygiiOrder: PerciformesFamily: Cichlidae Certified by: Leo Pascual Aquaculturist Phil-Fishgen, Freshwater Aquaculture Center, Central Luzon State University, Science City of Munoz, Nueva Ecija
12Genus: OreochromisSpecies: niloticusScientific name: Oreochromis niloticus The Nile tilapia (Oreochromis niloticus) was one ofthe first fish species cultured. Tilapia is more tolerantthan most commonly farmed freshwater fish to high salinity,high water temperature, low dissolved oxygen, and highammonia concentrations. In general, tilapia can survive inpH ranging from 5 to 10 but do best in a pH range of 6 to 9(Pompa and Masser, 1999). According to Khan, 2011, Nile Tilapia belongs to theFamily: Cichlidae (Cichlids and Tilapias), OrderPerciformes (Perch and Cichlids), and Class Actinopterygii(Ray-finned Fish).General Characteristics and Occurrence of Aeromonas sobriaveronii Plate 2.2: Pure culture Aeromonas sobria veroniiKingdom: BacteriaPhylum: ProteobacteriaClass: GammaproteobacteriaOrder: AeromonadalesFamily: Aeromonadacaea Certified by: Dr. Apolinario V. Yambot Head,Molecular Biology and Biotechnology Laboratory College of Fisheries Dean of college of Fisheries Central Luzon State University, Science City of Munoz, Nueva Ecija
13Genus: AeromonasSpecies: sobria veroniiScientific name: Aeromonas sobria veronii The Genus Aeromonads consists of straight,cocobacillary to bacillary, gram-negative bacteria withrounded ends. They occur in singly, in pairs and rarely asshort chains. Motile strains produce a single polarflagellum; some strain has petritrichous or lateralflagella. Aeromonas species is facultative anaerobic,catalase positive and oxidase positive. They grow optimallywithin a temperature range between 22-35 degree Celsius andtolerate a pH range from 4.5 to 9.0, but the optimum pHrange is from 5.5 to 9.0 (Hasan and Jaful, 2006). Aeromonads are primarily aquatic organisms occurringnaturally in different fresh water bodies that includerivers, water, streams and lakes. They are predominant inestuarine waters and easily isolated from seashore but notfrom deep sea. They also occur in raw sewage, treatedsewage and activated sludge (Ghenghesh, Ahmed, El-Khalek,Al-Gendy and Klena, 2008). Aeromonas species are belonging to the familyAeromonadaceae, that have a broad host spectrum both coldand warm blooded animals and are known as psychrophilic and
14mesophilic. Motile aeromonas were phenotipically classifiedinto the species Aeromonas hydrophila, Aeromonas sobria andAeromonas caviae (Aberoum and Jooyandeh, 2010). Aeromonas species can also be found in variousconcentrations of drinking water. The chronic exposure ofimmunocompromised persons to Aeromonas via contaminatedwaters could potentially lead to invasive disease, such assepticemia. Other studies have found aeromonads in dairyproducts (4%), vegetables (26% to 41%), and meats andpoultry (3% to 70%), with the largest numbers recorded forshellfish (31%) and fish (72%) (Janda and Abbott, 2010). Aeromonads are also belonging to the autochthonousflora of fishes and amphibians (Ashiru, Uaboi-Egbeni,Oguntowo, Idika, 2011).Pathogenicity of Aeromonas sobria veronii in Tilapia Bacterial infections, caused by motile members of thegenus Aeromonas, are among the most common and troublesomediseases of fish raised in ponds and recirculating systems.The disease caused by Aeromonas sobria occurs in all lifestages of Tilapia (Dabrowski, 2012). The widespreaddistribution of these bacteria in the aquatic environmentand the stress induced by intensive culture practicespredisposes fish to infections. Motile aeromonad infectionshave been recognized for many years and have been referred
15to by various names, including motile aeromonad septicemia(MAS), motile aeromonad infection (MAI), hemorrhagicsepticemia, red pest, and red sore. Aeromonas hydrophila, Aeromonas sobria, Aeromonascaviae, and possibly other aeromonads, is capable ofproducing disease in fish. Aeromonads are considered to beopportunistic pathogens, capable of producing disease onlyin weakened populations of fish or as secondary invaders infish suffering from other diseases (Camus, Durborow,Hemstreet, Thune and Hawke, 1998). Aeromonas sobria are probably one of the most commonbacterial diseases that infect wild and cultured tilapia.They cause fin rot or skin rot disease and may lead toheavy mortality in cultured tilapia (El-Sayed, 2006). In these instances some factor, usually stress hascaused the fish to become more susceptible to the bacteria.Common sources of stress are poor water quality,overcrowding or rough handling (Hossain, 2008). Infected fish frequently have small pinpointhemorrhages at base of the fins or on the skin, distendedabdomens and protruding eyes. Internal signs include thefluid in the abdomen, swollen liver and spleen and theintestine are distended and fluid filled (Floyd, R.F.,2009).
16 It was recently reported that epizootic ulcerative(EUS) caused by Aeromonas sobria resulted in great damageto fish farms in parts of southeast Asia such as Bangladeshand India (Aberoum and Jooyandeh, 2010). The role of aeromonads as a causative agent of fishdiseases has been known for decades, longer than theircomparable role in causing systemic illnesses in humans.The disease has several presentations, ranging from anacute form characterized by septicemia with accompanyinghemorrhages at the bases of fins, inappetence, andmelanosis to a subacute to chronic variety in older fish,consisting of lethargy, slight exophthalmia, andhemorrhaging in muscle and internal organs. Mesophilic species (Aeromonas hydrophila and Aeromonassobria veronii) causes also a similar assortment ofdiseases in fish, including motile Aeromonas septicemia(hemorrhagic septicemia) in carp, tilapia, perch, catfish,and salmon, red sore disease in bass and carp, andulcerative infections in catfish, cod, carp, and goby(Janda and Abbott, 2010).Chemical control of the bacteria in the field by way ofantibiotic Oxytetracycline (Terramycin) has been the drug ofchoice for treating motile aeromonad septicemias in fishes.
17The drug is approved for use with pond fishes, channelcatfish, and salmonids (Cipriano, 2001). The antibiotics are applied to fish in severalmethods. The usual preferred method is oral via medicatedfeed. More medicated feed are commercially prepared eithersinking of floating pellets. One of the problems with theuse of this method is that diseased fish stop eating thepellet and maybe compounded by unpalatable feed caused bythe presence of antibiotics which makes the problem worse. The more effective treatment than using medicatedfeed is through injection. It quickly leads to thebloodstream and tissues. But it is only practical forindividual fish rather than fish in a large scaleproduction. Open sores or ulcers can be treated by the use oftopical treatment such as iodine-based solution. This isusually necessary for more valuable individual fish such asornamental and brood stock. The method that can use for surface infections such asfin rot, bacterial gill disease and superficial fungalinfection is through Bath and Dip. Fish treatment by bathusually uses a separate container or tank and additionalaeration may also require for bath treatments (Rodgers andFurones, 2009).
18Advantages of using antibiotics According to Yanong (2003), tetracycline and otherrelative antibiotics are considered as a broad-spectrumantibiotics means that it is effective against a widevariety of bacteria. Tetracycline has been extensively used in the therapyof human and animal infections, for prophylactic purposesin animals and plants and for growth promotion in foodanimals also it is widely used in veterinary medicinemainly for treatment of skin bacterial infections. Thetarget animal species for the application of tetracyclinesare pig, cattle, sheep, goat and fish. Forms of tetracyclines permeate through the bacterialcell wall by passive diffusion and through the cytoplasmicmembrane. Antibacterial activity of typical tetracyclinesis associated with reversible inhibition of proteinsynthesis (Michalova, Novotna, and Schlegelova, 2004). According to Moellering and Standiford, (1990) ascited by Michalova, Novotna, and Schlegelova, (2004),Tetracyclines exhibit the activity against a broad spectrumof pathogenic microorganisms, exhibit low toxicity and arerelatively inexpensive. The antibiotics generally kills the bacteria byinterfering with either the formation of the bacterium cell
19wall, also it stops the bacteria from multiplication, DNAreplication or other aspects of bacterial cellularmetabolism, example include tetracycline, sulfonamides,chloramphenicol and macrolides (Romero, Feijoo, andNavarrete, 2012).Disadvantages of using antibiotics According to Yanong (2003), Tetracycline andoxytetracycline binds to magnesium and calcium renderingthem inactive. This means that with increasing waterhardness, it is necessary to increase the dosages requiredfor the drugs to have any effectiveness. Also antibiotics in and of themselves do not cure thefish instead they merely control the population of bacteriain a fish long enough for its immune system to eliminatethem. Some antibiotics like Tetracycline contribute to thepoor water quality and may be harmful to the fish since itis light sensitive and turn into brown when decomposing(Yanong, 2003). Due to antibiotic treatment there is a possiblemodification in gastrointestinal microbiota that couldalter this presumably beneficial host-microbiotarelationship. Antibiotic treatment can also eradicatemicroorganisms of the normal microbiota, facilitating theproliferation of opportunistic bacteria by depleting
20competition. One of the most commonly used antibiotics infish farms and hatcheries is Oxytetracycline, but it isvery poorly absorbed through the intestinal tract of fish.The use of oxytetracycline in fish farming has beendemonstrated to coincide with an increased frequency ofoxytetracycline-resistant microorganisms. The use of antimicrobial drugs in aquaculture has wellknown positive effects on the control of bacterialinfections; however several side effects that affect boththe fish and environment are associated with excessiveusage. Fish do not effectively metabolize antibiotics andwill pass them largely unused back into the environment infeces. It has been estimated that 75% of antibiotics fed tofish are excreted into the water (Romero, Feijoo andNavarrete, 2012).Taxonomic Classification and Morphological description:Jatropha curcas (Tuba-tuba) Plate 2.3: Jatropha curcas (Tuba-tuba)Kingdom: PlantaeDivision: MagnoliophytaClass: MagnoliopsidaOrder: MalpighialesFamily: Euphorbiaceae Certified by: Wilfredo F. Vendivil Ph. D. Curator II Botany Division
21Genus: JatrophaSpecies: curcasScientific name: Jatropha curcas The Jatropha curcas with a common name of Tuba-tubabelongs to Family Euphorbiaceae. It is a small tree growingas high as 9”. Leaves are alternate, cordate and 3-5 cutlobed. Flowers are yellowish-green, monoecious, in terminalumbels, staminate and pistillate flowers mingled withoutorder. Staminate: Calyx, 5 unequal sepals; Corolla bellshaped, 5 petals, woolly within, a small notch at the end,bent downward; stamens 10, in 2 whorls of 5. Pistillate:Calyx and Corolla as above; several tongue-like staminodesreplace the stamens; ovary free, oblong, 3-celled, 1 ovulein each cell; style 3 branched. Seed vessel: fleshy of 3capsules, each bearing 1 oval coriaceous seed (De Taveraand Pardo, 2000).Tinospora crispa (Makabuhay) Plate 2.4: Tinospora crispa (Makabuhay)Kingdom: PlantaeDivision: EquisetophytaClass: EquisetopsidaOrder: RanunculalesFamily: Menispermaceae Certified by: Wilfredo F. Vendivil Ph. D. Curator II Botany Division
22Genus: TinosporaSpecies: crispaScientific name: Tinospora crispa The Tinospora crispa with a common name of Makabuhaybelongs to Family Menispermaceae (De Tavera and Pardo,2000). It is a woody, climbing, vines or rarely erectshrubs or small trees with alternate, exstipulate, usuallysimple, pelate leaves, inflorescence chymes or panicles,rarely solitary. Flowers are small, green or whiteunisexual or dioeciously. Fruit is 1 seeded succulentdrupe. Seeds albuminous often curved like a horse shoe.Plant usually contain tonic, narcotic poisonous or bitterprinciple (Diguangco and Jose, 1950).Antimicrobial agents in Plants The group of compounds found in plants such asphenols, phenolic acids, quinones, flavones, flavonoids,flavonols, tannins and coumarins show antimicrobial effectand serves as plant defense mechanisms against pathogenicmicroorganisms. Plants synthesized flavones, flavonoids and flavonolsin response to microbial infection and are often foundeffective in vitro as antimicrobial substance against awide array of microorganisms.
23 Tannins possessed an astringent property. On the otherhand, Coumarins have a characteristic odor andantimicrobial properties. Fragrance of plant is carried by essential oilfractions which are secondary metabolites. Essential oilsalso posses’ strong antimicrobial properties. Naturallyfound alkaloids are commonly found to have antimicrobialproperty (Das, Shrivastava and Tiwari, 2010). Secondary metabolites present in plants have beenlinked with the healing properties of plants. In additionto their active ingredients, plants contain minerals,vitamins, volatile oils, glycosides, alkaloids,bioflavonoids, and other substances that are important insupporting a particular herbs medicinal properties thatplay a role in wound healing for instance, encourage bloodclotting, fight infections and accelerate the wound healingprocess ( Obi, Nwanebu, Ndubuisi-Nnaji, Onuoha andChiegboka, 2011). According to Krishnaiah et al., 2009 cited by Namuli,Abdullah, Sieo, Zuhainis and Oskoueian 2011, Plantsecondary metabolites (alkaloids, terpenoids and phenoliccompounds) are potential antimicrobial agents that can helpto alleviate problem of antibiotic resistance.
24Chemical compositions of:Jatropha curcas (Tuba-tuba) The leaf and bark of Jatropha curcas or Tuba-tuba havebeen shown to contain glycosides, tannins, phytosterols,flavonoids and steroidal sapogenins also it is used intreating skin infections and wound (Esimone, Nworu andJackson, 2009). The leaves of Jatropha curcas contain flavonoids,apigenin, vitexin, isovitexin, dimer of atriterpenes and 2-flavonoidal glycosides (Islam, Yaakob and Anuar, 2011). Igbinosa et al. (2009) and Akinpelu et al. (2009)observed the presence of saponins and Flavonoids inJatropha curcas (Tuba-tuba) stem barks and leaves extractrespectively. Also the presence of gallic acid has alsobeen reported in the leaves extract according to Manpong etal., (2009). According to Diwani et al., (2009) and Manponget al., (2009), leaves of Jatropha curcas plant containphenolic compounds such as Corilagin and ellagic acid. AlsoSubramanian et al., (1971), state that present studies havebeen reported the compounds apigenin, vitex, and isovitexin the leaves of Jatropha curcas, these are all are citedby Namuli, Abdullah, Sieo, Zuhainis and Oskoueian, 2011.
25 Vanillic acid, a form of benzoic acid, was observed tobe present in the methanol extract of leaves and root barks(Namuli, Abdullah, Sieo, Zuhainis and Oskoueian, 2011).Chemical composition of Tinospora crispa (Makabuhay) Tinospora crispa (Makabuhay) contains a bitterprinciple colombine, traces of alkaloid, and a glucoside.The bitter principle is glucosidal in nature. Also itcontains diterpenes which has an antimicrobial effect(Cruz, 2000).Related Studies According to Haas et al., (2002) as cited by Ahmed andSalimon, (2009), observed six phorbol esters from Jatrophacurcas. The seeds and seed oil are toxic to human and animalsand for nutritional utilization are not possible. Phorbolesters have been identified as the major toxic compounds inJatropha curcas. The biological effect of these compoundsbrings about a wide range of biochemical and cellulareffects, alter cell morphology, serve as lymphocytemitogens and induce platelet aggregation. The toxic fraction isolated from Jatropha curcas oilnot only had an irritant effect after topical applicationbut also caused diarrhea and mortality in the animals,indicating that there is a substantial percutaneous
26absorption of the toxic components of the oil (Ahmed andSalimon, 2009). Diterpenes are believed to be the most potentcompounds synthesized by Jatropha curcas. Phorbol estersare the most toxic molecules in Jatropha and the sixphorbol esters were identified in Jatropha curcas. Thesensitivity of toward toxic constituents in Jatropha plantextracts varies with different fish. Leaf extracts were nontoxic up to 5 g/L. The degree of toxicity actually varieswith extract types, nature of test substances, dose, andmode of administration and sensitivity of animals (Becker,Klaus, Makkar, Harinder, Devappa and Rakshit, 2012). Makabuhay has antibacterial property because itcontains diterpenes which was recorded to be effectiveagainst Psuedomonas aeruginosa, a gram-negative bacterium(Cruz, 2000). According to Eisa et al. (1994), as cited by Cipriano,(2001), shown that the prevalence of Motile AeromonadSepticemia in cultured and wild Nile tilapia (Oreochromisniloticus) was 10.0% and 2.5% respectively; it was 18.75%and 6.25% in cultured and wild Karmout catfish,respectively. In the study of Evaluation of Botanical piscicides onNile Tilapia Oreochromis niloticus and Mosquito fish
27Gambusia affinis, by Cagauan, Galaites and Fajardo, 2004,pointed out that the toxic parts of plants employed as fishpoisons includes the roots, seeds, fruits, bark, latex orleaves. The toxicity of Jatropha curcas or physic nut(Tuba-tuba) to Nile tilapia is 12.8 mL l concentration.Based on the 24-hour lethal concentration (LC100), theplants with the strongest piscicidal activity to Niletilapia and mosquito fish were Makabuhay with 0.82 mL 1-1toxic concentration. According to Ashiru, Uaboi-Egbeni, Oguntowo and Idika,2011, The Aeromonas species exhibited different level ofantibiotics susceptibility based on the zone of inhibitionobserved around the antibiotics disc. Aeromonas caviae,Aeromonas sobria and Aeromonas hydrophila were allresistant to tetracycline, nitrofurantoin and augmentinwith an average zone of inhibition of 9mm, 10mm and 8mmrespectively. In the study of Ulcerative Aeromonas Infections inTilapia (Cichlidae: Tilapiini) from Mtera Hydropower Dam,Tanzania, Shayo SD, Mwita CJ and Hosea K, 2012, Aeromonadinfections are certainly the causative agent of theulcerative fish disease outbreaks in Mtera Dam, Tanzania.Most of the isolates from the infected fish showed typicalaeromonad characteristics. The fishes had hemorrhages at
28the base of the fins and/or on the skin, and grossulcerative lesions. Internal signs included, fluid in theabdomen, swollen liver and spleen and fluid-filleddistended intestine. According to Ibrahem, Mostafa, Arab and Rezk, 2008, intheir study Prevalence of aeromonas hydrophila infection inwild and cultured tilapia Nilotica (Oreochromis niloticus)in Egypt revealed that Aeromonas hydrophila infection, thecause of the Motile Aeromonas Septicemia (MAS), has adifferent seasonal distribution within the wild and thecultured Oreochromis niloticus. The observed clinical signsin the examined fish suffering from Motile AeromonasSepticemia (MAS) were previously reported by Okpkowassiliand Okpkowassili (1994); Viola, (1995) and Ali, (1996) whoreported that septicemia, ascitis, erosion, ulceration,detachment of scale, exophthalmia and muscular necrosis arethe most predominant clinical signs of MAS in Nile tilapia. According to Rahman, Akanda, Rahman, and Chowdhury,2009, in their study Evaluation of the efficacies ofselected antibiotics and medicinal plants on commonbacterial fish pathogens, Crude extracts were prepared fromvarious parts (leaves and bulb) of garlic, turmeric, akandand neem and four different doses were applied to the freshculture of pathogenic isolates under the in-vitro
29condition. Finding out those medicinal plants would be aneffective control measure along with antibiotics againstbacterial fish diseases. In the study of Haniffa and Kavitna, 2012, entitledAntibacterial activity of medicinal herbs against the fishpathogen Aeromonas hydrophila, the antagonistic effect ofthe methanolic extracts of Coleus aromaticus (Oregano) ofLamiaceae and Tabernaemontana divaricata of Apocynaceaewere found to be most effective against Aeromonashydrophila. Considering overall performance, Coleusaromaticus was found to be the most effective antagonisticagent against Aeromonas hydrophila.Analysis of the Related Literature and Related Studies The related literature and related studies citedcontained information relevant to the experiment. Variousscientific ideas and results are presented in order toprovide integral approach towards the antibacterialactivity of Aeromonas sobria. Aeromonas sobria and other motile aeromonads arecapable of causing disease to Nile Tilapia (Oreochromisniloticus) and other fishes. The diseases caused byAeromonads are commonly known as Motile AeromonasSepticemia. Skin lesions, hemorrhages, tail and fin rots
30are one of the symptoms of MAS (Motile AeromonadsSepticemia). Administering antibiotics such as Tetracycline andTerramycin in the form of medicated pellets are the mostcommon treatments of disease caused by Aeromonads and otherfish diseases. However due to the development of antibioticresistant on fish pathogen and side effect on theenvironment, researchers finds new antimicrobial agents outof plants or medicinal plants that can be an alternativefor Tetracycline and other antibiotics. In the study of Haniffa and Kavitna, 2012, entitledAntibacterial activity of medicinal herbs against the fishpathogen Aeromonas hydrophila, the antagonistic effect ofthe methanolic extracts of Coleus aromaticus of Lamiaceaeand Tabernaemontana divaricata of Apocynaceae were found tobe most effective against Aeromonas hydrophila. From all the test plants used by different researches,Jatropha curcas (Tuba-tuba) and Tinospora crispa(Makabuhay) were included in the list of test plantsrecognized with potentials as antibacterial agents. Jatropha is proven to have antimicrobial activityagainst gram-negative bacteria, Pseudomonas aeruginosawhile Makabuhay also contains antibacterial property due to
31presence of diterpenes which was recorded to be alsoeffective against Pseudomonas aeruginosa. Aeromonas sobria has been associated with diseases offish including Tilapia; it causes fin and tail rots orulcer disease in fishes. Remedies against bacterialinfections are highly needed because some strain ofAeromonas are resistant against antibiotics. Therefore, adevelopment of alternative antimicrobial drugs frommedicinal plants such as Jatropha curcas (Tuba-tuba) andTinospora crispa (Makabuhay) is necessary for the treatmentof infectious disease cause by the Aeromonas sobria. After a thorough review of the related literature andstudies, it was found out that there was no study which hasbeen conducted on the antimicrobial activity of the leaveextracts of Jatropha curcas (Tuba-tuba) and Tinosporacrispa (Makabuhay) against Aeromonas sobria in Tilapia (Oreochromis niloticus).
32 CHAPTER III METHODOLOGY This chapter presents the kind of research andresearch design used for the study as well as theprocedures of the experiment. The procedures used in thisstudy was based on the study conducted by Cagauan,Galaites, and Fajardo, regarding the Evaluation ofBotanical Piscicides on Nile Tilapia and Mosquito Fish(2004).Kind of Research The kind of research used in determining theantibacterial activity of Jatropha curcas (Tuba-tuba) andTinospora crispa (Makabuhay) against the fish pathogenAeromonas sobria was experimental research. An experimentalresearch is a research wherein researchers manipulates andcontrols one or more independent variables for variationconcomitant to manipulation of independent variables. Inthis study, the independent variable was the extract of theleaves of Jatropha curcas (Tuba-tuba) and Tinospora crispa(Makabuhay) and the dependent variable was theantimicrobial activity of the plants. While the extraneousvariables were the amount and kind of water each basincontained, the number of fishes per basin, and also thenumber of days of toxicity test.
33Research Design The study was divided into two parts, the toxicitytest and antimicrobial assay and both used randomizedcomplete block design. In toxicity test, the test plantswere the leaves of Jatropha curcas (Tuba-tuba) andTinospora crispa (Makabuhay). The subjects were placed indifferent experimental unit completely at random; therewere 2 concentrations in each plant sample, in Jatrophacurcas (Tuba-tuba) the concentrations used were 0.13% and0.11%, while in Tinospora crispa (Makabuhay) theconcentrations used were 0.0045% and 0.0040%. Eachconcentration was replicated three times to determine thecause of change. The control variable was water and thetest plants undergo randomization process. Also, intoxicity test, the blocks were divided into 5 blocks whilein antimicrobial assay; it was divided into 6 blocksshowing the number of concentration of each plant andcontrol setup. (See figure 3.1 and 3.2)Figure 3.1: Toxicity testLegend:I- Jatropha curcas (Tuba-tuba) IA- 0.13% IB- 0.11%II- Tinospora crispa (Makabuhay) IIA- 0.0045% IIB- 0.0040%III- Control IIIA- 0%
34 IIIA3 IIB1 IA2 IIA3 IIB3 IB2 IIA1 IIIA1 IA3 IB3 IB1 IIA2 IIIA2 IA1 IIB2 Trials or Replication (1, 2, and 3)Figure 3.2: Antimicrobial assayDesign for the arrangement of disk in a petridish forJatropha curcasLegend: I- Jatropha curcas (Tuba-tuba) A. 0.13% B. 0.11% C. 0.09% D. 0.07% E. 0.05% 2 1 2 1 2 1 IA IB IC 3 3 3 2 1 2 1 ID IE 3 3 Trials or Replication (1, 2, and 3)
35Design for the arrangement of disk in a petridish forTinospora crispaLegend: II- Tinospora crispa (Makabuhay) A. 0.0045% B. 0.0040% C. 0.0035% D. 0.0030% E. 0.0025% 2 1 2 1 2 1 IIA IIB IIC 3 3 3 2 1 2 1 IID IIE 3 3 Trials or Replication (1, 2, and 3)
36Design for the arrangement of disk in a petridish forTetracycline (Positive control)Legend: III- Tetracycline a. 0.13% f. 0.0045% b. 0.11% g. 0.0040% c. 0.09% h. 0.0035% d. 0.07% i. 0.0030% e. 0.05% j. 0.0025% 2 1 2 1 2 1 IIIa IIIb IIIc 3 3 3 2 1 2 1 2 1 IIId IIIe IIIf 3 3 3 2 1 2 1 2 1 IIIg IIIh IIIi 3 3 3 2 1 IIIj 3 Trials or Replication (1, 2, and 3)
37Figure 3.3: Flowchart of the Study Collection of leaves of Jatropha curcas (Tuba- tuba) and Tinospora crispa (Makabuhay) Aqueous extraction of leaves of sample plants Dilution of stock solution in different concentration for Jatropha curcas (0.13%, 0.11%, 0.09%, 0.07%, and 0.05%) and for Tinospora crispa (0.0045%, 0.0040%, 0.0035%, 0.0030%, and 0.0025%). Experimental set-up for toxicity test Selection of Tilapia fingerlings Acclimatization of Nile Tilapia Fingerlings Test of Oreochromis niloticus (Tilapia) fingerlings in different concentrations Analysis of data and Determination of Lethal concentration Sterilization of materials Preparation of Culture medium (Mueller-Hinton agar) Inoculation of Aeromonas sobria Incubation of Aeromonas sobria Measurement of zone of inhibition Proper wastes disposal Gathering and Analysis of Data Statistical Analysis of Data
38 MATERIALS AND METHODS OF THE STUDYCollection of Plant Materials Three hundred fifty grams (350 g) of Jatropha curcas(Tuba-tuba) and Tinospora crispa (Makabuhay) were freshlycollected from Urbanville Talon 3 Las Pinas City. Theleaves of the plant samples were used for aqueousextraction.Preparation of Extracts of Tuba-tuba and Makabuhay leaves The leaves of plant samples were separated manuallyand were cleaned with sterile distilled water. The leaveswere weighed using a spring balance to get the wet weight.After getting the wet weight the leaves were sun dried. Thedried leaves were weighed again to get the dry weight. For aqueous extraction, the dried leaves were finelyground using a blender and the 100 g of powdered leaveswere macerated and soaked in 200 ml of distilled water. The extracts were filtered through Whatman filterpaper No. 1 and were squeezed through 4 layers of cloth.And the aqueous extracts of plant samples were freeze driedfor storage. The filtrate obtained was diluted in distilled waterto give the desired concentrations; for Jatropha curcas(0.13%, 0.11%, 0.09%, 0.07%, and 0.05%) and for Tinospora
39crispa (0.0045%, 0.0040%, 0.0035%, 0.0030%, and 0.0025%).The different plant extract concentrations were stored insterile bottles and were kept in the refrigerator and wereused for toxicity test and antimicrobial tests.Toxicity TestExperimental set up The water from Philippine Normal University was usedfor acclimatization and toxicity test. Also, the water wasstocked for two days to separate the chlorine content ofthe water. The aquarium, with aerator, was used in acclimatizingthe Nile Tilapia fingerlings (Oreochromis niloticus).Plastic basin containers with net covers were used intoxicity test and each container was filled with 1000 mlwater. The water was pre-aerated for 40 minutes to fulloxygen saturation before the different volumes of the plantextract was added. For each plant there were two differentconcentrations and 0 ml/L for control set up, each werereplicated three times.Selection of Tilapia fingerlings Fish species Oreochromis niloticus (Tilapia) with thesize of 1 cm in length and with the weight of 0.1 to 0.5
40grams were obtained from Phil-Fishgen, FreshwaterAquaculture Center, Central Luzon State University, ScienceCity of Munoz, Nueva Ecija.Acclimatization of Nile Tilapia Fingerlings All Fingerlings were exposed to water (non chlorinatedtap water) in an aquarium for 30 minutes before they weresubjected to toxicity test. The aquarium was covered duringthe holding period to minimize stressing the fish due toactivity outside the holding aquarium. When mortalityoccurred, the dead fishes were removed and the healthy/live fishes were selected in subjecting to toxicity test.Test of Oreochromis niloticus (Tilapia) fingerlings Proper care was observed to minimize stress incurredby the test fish. Ten Nile tilapia fingerlings were stockedper basin after the addition of the plant extract. The different concentrations of the plant sample wereadded to the non-chlorinated water of each basin container,the concentration of plant samples are the following: forJatropha curcas (Tuba-tuba) (0.13% and 0.11%) and forTinospora crispa (Makabuhay) (0.0045% and 0.0040%), eachconcentration was replicated 3 times. In addition, acontrol set-up was made wherein non-chlorinated water was
41added to each basin container, with 0 mL/L concentration,and was replicated 3 times. The selected healthy fisheswere transferred from the holding aquarium to each basinusing a scoop net. The netting was not allowed to contactthe test solution to prevent contamination to fish in theholding aquarium. The time was recorded when the fish wastransferred into the basin containers. This was the timewhen the test was monitored for four days (24, 48, 72 to 96hours). The fish mortalities was observed and recorded fromstocking every 24 hours for four days. The dead fishes wereremoved immediately. A fish is considered dead when it doesnot respond to mechanical prodding. The temperature, dissolved oxygen, and pH of the waterwere monitored initially after the addition of plantextract, before fish stocking and 24 hours thereafter. The fish mortalities were observed and recorded at 24,48, 72 and 96 hours from stocking. In addition, staticprocedure test was employed which means that the solutionsremained unchanged throughout the duration of the test.Analysis of data and Determination of Lethal concentration The lethal concentrations of the test plant, Jatrophacurcas (Tuba-tuba) and Tinospora crispa (Makabuhay) wasdetermined by plotting concentrations of the plant against
42fish mortality within 24 hours, 48 hours, 72 hours, and 96hours after exposure to the treatment. The determination ofLethal concentration 50 (LC50) was determined throughProbit Analysis.Sterilization of Materials All the materials and glassware, inoculatinginstruments, Mueller-Hinton agar and EC broth weresterilized using an autoclave with the standard of 121degree Celsius at 15 psi for 30 minutes. All the proceduresin the subculture test and susceptibility test of organismwas performed aseptically to avoid any contamination of theculture and materials.Preparation of Tetracycline The Tetracycline solution was prepared bydissolving 500 mg of tetracycline capsule to 500 mL ofdistilled water.Preparation and sterilization of filter paper discs Whatman filter paper no. 1 was used to prepare discsapproximately 6 mm in diameter which was made using a paperpuncher. The prepare discs were sterilized in an autoclaveat 15 psi for 30 minutes. The sterilized discs were soakedinto the aqueous extracts of plant samples and tetracyclinefor 1 hour before dispensing. The different concentrations
43of aqueous extracts of leaves of Jatropha curcas (Tuba-tuba) and Tinospora crispa (Makabuhay) served as thetreatments and tetracycline was used as the positivecontrol.Preparation of Culture medium (Mueller-Hinton agar) In a beaker, 19 g of the medium was suspended in500mL of distilled water. Using hot plate, the medium washeated with frequent agitation and was boiled for 1 minuteto completely dissolve the medium. After that, the mediumwas sterilized using an autoclave at 121 degrees Celsiusfor 15 minutes and was cooled to room temperature. Thecooled MH agar was poured to the sterilized Petri dish.Approximately ¾ of the Petri dish was filled with melted MHagar. The melted MH agar was allowed to cool at roomtemperature to solidify. Also 7.4 grams of EC broth wassuspended in 200 mL of distilled water.Inoculation of Aeromonas sobria veronii The pure culture of Aeromonas sobria veronii wasobtained from the Molecular Biology and BiotechnologyLaboratory College of Fisheries, Central Luzon StateUniversity, Science City of Munoz, Nueva Ecija.
44 Pour plate technique was used in the study. From thepure culture, at least three to five well-isolated colonieswas selected from an agar plate culture and was transferredinto a tube containing 10 mL of sterilized EC broth and wasshaken vigorously. Using a sterilized pipette, 1 mL wasremoved from the 10 mL or 100 culture tube and was placedinto the test tube containing 9 mL of sterilized EC brothor 10-1 tube and shaken vigorously. Using the same pipette,1 mL was removed from the 10-1 tube and was placed into the10-2 tube also containing 9 mL sterilized EC Broth andshaken again vigorously. This was repeated until reachedthe 10-5 tube. Using pipette, 1 mL of 10-5 culture tube wasplaced in the empty sterile petri dish, after that, meltedMueller Hinton agar was poured over it quickly, before theagar cooled, the plate was gently rotated in circularfashion to disperse the inoculum. Using sterile forceps, the impregnated discs wasplaced on the surface of the inoculated plate. The discswere positioned such that the minimum center distance is 24mm and no closer than 10 to 15 mm from the edge of thepetri dish.
45Incubation of Aeromonas sobria veronii In inverted position, the plate was incubated at roomtemperature (37 °C) for 24 to 48 hours.Measuring of Zone of Inhibition After 24 to 48 hours, using a ruler, the diameter ofzone of inhibition was measured in millimeter which was heldon the back of the inverted petri plate.Proper wastes disposal Culture plates, agar plates or test tubes that havebeen used to grow micro-organisms were subjected in anautoclave or pressure cooker at 121 degrees Celsius for 15minutes. After sterilizing, the culture media from reusablepetri dishes and test tubes were removed and discarded intoa container. The reusable petri dishes and test tubes werewashed and dry. On the other hand, the disposable petridishes were discarded into the same container mentionedabove.
46 CHAPTER IV RESULTS AND DISCUSSION This chapter presents the data gathered from toxicitytest and antibacterial assay as well as the statisticalanalysis and interpretations of data. The first sub-problem of this study was to determinethe mortality rate of Tilapia fingerlings in 0.13% and0.11% concentration of Jatropha curcas (Tuba-tuba) andTinospora crispa (Makabuhay) with the concentration of0.0045% and 0.0040% while 0 % for negative control.Table 4.1 Summary of the average mortality rate of NileTilapia Fingerlings BLOCK HOURS 24 48 72 96 Total % mortality IA 0 0 1 1 2 20 IB 0 0 0 1 1 10 IIA 0 0 1 1 2 20 IIB 0 0 0 1 1 10 IIIA 0 0 0 0 0 0
47Figure 4.1 Graph of the percent mortality rate of NileTilapia Fingerlings on 24, 48, 72 and 96 hours of exposureto different test plant Mortality rate of Nile Tilapia Fingerlings 1 number of dead fish 0.8 0.6 IA IB 0.4 IIA 0.2 IIB 0 IIIA 24 48 72 96 Time of Exposure (Hours)Legend: I- Jatropha curcas (Tuba-tuba) A- 0.13% B- 0.11% II- Tinospora crispa (Makabuhay) A- 0.0045% B- 0.0040% III- Negative Control A- 0 % Table 4.1 shows the summary of the mortality rate ofNile Tilapia fingerlings (Oreochromis niloticus) after 24,48, 72 and 96 hours of exposure to water with different
48concentration of test plants (Jatropha curcas and Tinosporacrispa) and water. After 24 and 48 hours of Toxicity test,the recorded mortality was 0 in all the concentrations ofthe Tuba-tuba (0.13% and 0.11%) and Makabuhay (0.0045% and0.0040%) including the negative control. Moreover, therewas an increased mortality rate of Nile Tilapia fingerlingsafter 72 and 96 hours of exposure to the differentconcentrations of Tuba-tuba and Makabuhay plant extracts.The graph (Figure 4.1) above illustrated the saidobservation. The recorded mortality of fingerlings after 72 hoursof exposure of fingerlings in Tuba-tuba and Makabuhayhaving the concentration of 0.13% and 0.0045% was 1fingerling and after 96 hours of exposure, there was 1fingerling died. On the other hand, there was no mortalityrate recorded in the control set-up after 96 hours. Thetotal and percent mortality rate of Nile Tilapiafingerlings after 96 hours of exposure to Tuba-tuba plantwith the concentration of 0.13% is 2 fingerlings or 20percent and 1 fingerling or 10 percent for 0.11%concentration while for Makabuhay plant with theconcentration of 0.0045% the total mortality is 20 percentor 2 fingerlings and 10 percent or 1 fingerling for 0.0040%
49concentration and 0 percent for negative control (Table4.1).Summary of Toxicity Test In addition to the mortality rate of the Nile TilapiaFingerlings, the lethal concentration 50 (LC50) was alsocomputed wherein it determined the concentration of thetest plant that was lethal to the 50 percent of the testpopulation (Tilapia Fingerlings) after 96 hours ofexposure. The LC50 was calculated by the used of Probitanalysis and Linear regression using Microsoft excel. The computed lethal Concentration 50 of Jatrophacurcas (Tuba-Tuba) was 0.179 %, it only means that theconcentration of Tuba-tuba which is 0.13% and 0.11% are nottoxic or lethal to the Nile Tilapia Fingerlings. ForTinospora crispa (Makabuhay), the computed LC50 was 0.0056% which is lethal to the fingerlings therefore theconcentrations of Makabuhay which is 0.0045% and 0.0040%were also not toxic to Nile Tilapia fingerlings. Incomparison with the result of the study of Cagauan,Galaites, and Fajardo, in 2004, regarding the LC50 of tuba-tuba and makabuhay, for tuba-tuba, their computed lethalconcentration 50 was 1.28% which is higher than thecomputed LC50 we computed while for makabuhay, it was 0.082which is also higher than our computed LC50 for makabuhay.
50 The second sub-problem of this study was to determinethe zone of inhibition of Jatropha curcas (Tuba-tuba) andTetracycline with the concentrations of 0.13%, 0.11%,0.09%, 0.07% and 0.05%; Tinospora crispa (Makabuhay) andTetracycline with the concentrations of 0.0045%, 0.0040%,0.0035%, 0.0030% and 0.0025%.Table 4.2 Summary of means of Zone of Inhibition ofJatropha curcas (Tuba-tuba) and Tetracycline Zone of inhibition (mm) Mean Concentrations Jatropha curcas Tetracycline 0.13% 15.3 45.3 0.11% 14.6 41.6 0.09% 13 39.3 0.07% 0 38 0.05% 0 35.3Table 4.3 Summary of means of Zone of Inhibition ofTinospora crispa (Makabuhay) and Tetracycline Zone of Inhibition (mm) Concentrations Mean Tinospora crispa Tetracycline 0.0045 % 18.6 35.3 0.0040 % 16 28.60.0035 % 14.3 27.6
51 0.0030 % 13.6 27 0.0025 % 12 26.3 The table above shows the means of the zone ofinhibition of both Jatropha curcas (Tuba-tuba) andTetracycline (Table 4.2) and Tinospora crispa (Makabuhay)and Tetracycline (Table 4.3). Each concentration wastreated three (3) times. The decrease in the measurement ofzone of inhibition was observed as the concentration ofboth Jatropha curcas, Tinospora crispa and Tetracyclinegets lower. The third sub-problem of this study was to determineif there is significant differences among the zones ofinhibition of Jatropha curcas (Tuba-tuba) and Tetracyclinewith the concentrations of 0.13%, 0.11%, 0.09%, 0.07% and0.05%; Tinospora crispa (Makabuhay) and Tetracycline withthe concentrations of 0.0045%, 0.0040%, 0.0035%, 0.0030%and 0.0025%.
52Table 4.4 ANOVA: Zone of Inhibition of Jatropha curcas(Tuba-tuba) and Tetracycline Sum of Mean Squares Df Square F Sig. Remarks Between HIGHLY 1833.2 9 Groups SIGNIFICANT 203.6889 34.9181 0.0000 Within 116.6667 20 Groups 5.833333 Total 1949.867 29 The table above is an Analysis of Variance (ANOVA)which shows the results of the comparison among the meansof the zones of inhibitions of different concentrations ofJatropha curcas (Tuba-tuba) and Tetracycline. The NullHypothesis (Ho) is rejected, due to that there issignificant differences among the computed means oraverages of the observed values from each concentration,since the p-value is equal to 0.0000 and is less than thelevel of significance (α), p value (Sig.) = 0.0000 < α =0.05. It implies that we are 95% confident to say that themean effects of the five (5) concentrations havedifferences from each other. It only means that among thefive (5) concentrations of Tuba-tuba that will be used or
53be applied have different effects to Aeromonas sobriaveronii.Table 4.5 ANOVA: Zone of Inhibition of Tinospora crispa(Makabuhay) and Tetracycline Sum of Mean Squares Df Square F Sig. Remarks Between HIGHLY Groups 9 SIGNIFICANT 8283.8667 920.4296 110.8951 0.0000 Within 20 Groups 166 8.3 Total 8449.8667 29 The table above is an Analysis of Variance (ANOVA)which shows the results of the comparison among the meansof the zones of inhibitions of different concentrations ofTinospora crispa (Makabuhay) and Tetracycline. The NullHypothesis (Ho) is rejected, due to that there issignificant differences among the computed means oraverages of the observed values from each concentration,since the p-value is equal to 0.0000 and is less than thelevel of significance (α), p value (Sig.) = 0.0000 < α =0.05. It implies that we are 95% confident to say that themean effects of the five (5) concentrations havedifferences from each other. It only means that among the
54five (5) concentrations of Makabuhay that will be used orbe applied have different effects to Aeromonas sobriaveronii. The fourth sub-problem of this study is to determinethe best leaf extract among Jatropha curcas and Tinosporacrispa.Table 4.6 Post Hoc Test on Zone of Inhibition of Jatrophacurcas Pair Mean Critical Remarks Difference value IA-IB 0.7 8.3 NOT SIGNIFICANT IA-IC 2.3 8.3 NOT SIGNIFICANT IA-ID 15.3* 8.3 SIGNIFICANT IA-IE 15.3* 8.3 SIGNIFICANT IA-IIIa 30* 8.3 SIGNIFICANT IA-IIIb 26.3* 8.3 SIGNIFICANT IA-IIIc 24* 8.3 SIGNIFICANT IA-IIId 22.7* 8.3 SIGNIFICANT IA-IIIe 20* 8.3 SIGNIFICANT IB-IC 1.6 8.3 NOT SIGNIFICANT IB-ID 14.6* 8.3 SIGNIFICANT IB-IE 14.6* 8.3 SIGNIFICANT
56 IIIa-IIIb 3.7 8.3 NOT SIGNIFICANT IIIa-IIIc 6 8.3 NOT SIGNIFICANT IIIa-IIId 7.3 8.3 NOT SIGNIFICANT IIIa-IIIe 10* 8.3 SIGNIFICANT IIIb-IIIc 2.3 8.3 NOT SIGNIFICANT IIIb-IIId 3.6 8.3 NOT SIGNIFICANT IIIb-IIIe 6.3 8.3 NOT SIGNIFICANT IIIc-IIId 1.3 8.3 NOT SIGNIFICANT IIIc-IIIe 4 8.3 NOT SIGNIFICANT IIId-IIIe 2.7 8.3 NOT SIGNIFICANT The table above shows the multiple comparison of themeans of the different concentrations of Jatropha curcas(Tuba-tuba) and Tetracycline. (*) The mean difference aboveis significant at the .05 level. This means that there is asignificant difference in the zone of inhibition betweenthese pairs. The positive control or Tetracycline, IIIa,IIIb, IIIc, IIId and IIIe with the concentration of 0.13%,0.11%, 0.09%, 0.07 % and 0.05 % respectively have a greaterzone size (mm) compare to the different concentrations ofTuba-tuba (IA- 0.13%, IB- 0.11%, IC- 0.09%, ID- 0.07%, andIE- 0.05%). For the other pairs the mean differences arenot significant.
57 Table 4.7 Post Hoc Test on Zone of Inhibition ofTinospora crispa Pair Mean Critical Remarks Difference value IIA-IIB 2.6 8.3 NOT SIGNIFICANT IIA-IIC 4.3 8.3 NOT SIGNIFICANT IIA-IID 5 8.3 NOT SIGNIFICANT IIA-IIE 6.6 8.3 NOT SIGNIFICANT IIA-IIIf 13* 8.3 SIGNIFICANT IIA-IIIg 10* 8.3 SIGNIFICANT IIA-IIIh 9* 8.3 SIGNIFICANT IIA-IIIi 9* 8.3 SIGNIFICANT IIA-IIIj 8.4* 8.3 SIGNIFICANT
58IIB-IIC 1.7 8.3 NOT SIGNIFICANTIIB-IID 2.4 8.3 NOT SIGNIFICANTIIB-IIE 4 8.3 NOT SIGNIFICANTIIB-IIIf 15.6* 8.3 SIGNIFICANTIIB-IIIg 12.6* 8.3 SIGNIFICANTIIB-IIIh 11.6* 8.3 SIGNIFICANTIIB-IIIi 11.6* 8.3 SIGNIFICANTIIB-IIIj 11* 8.3 SIGNIFICANTIIC-IID 0.7 8.3 NOT SIGNIFICANTIIC-IIE 2.3 8.3 NOT SIGNIFICANTIIC-IIIf 17.3* 8.3 SIGNIFICANTIIC-IIIg 14.3* 8.3 SIGNIFICANTIIC-IIIh 13.3* 8.3 SIGNIFICANT
60 IIIf-IIIi 4.6 8.3 NOT SIGNIFICANT IIIf-IIIj 5.3 8.3 NOT SIGNIFICANT IIIg-IIIh 1 8.3 NOT SIGNIFICANT IIIg-IIIi 1.6 8.3 NOT SIGNIFICANT IIIg-IIIj 2.3 8.3 NOT SIGNIFICANT IIIh-IIIi 0.6 8.3 NOT SIGNIFICANT IIIh-IIIj 1.3 8.3 NOT SIGNIFICANT IIIi-IIIj 0.7 8.3 NOT SIGNIFICANT The table above shows the multiple comparison of themeans of the different concentrations of Tinospora crispa(Makabuhay) and Tetracycline. (*) The mean difference aboveis significant at the .05 level. This means that there is asignificant difference in the zone of inhibition between
61these pairs. The positive control or Tetracycline, IIIf,IIIg, IIIh, IIIi and IIIj with the concentration of0.0045%, 0.0040%, 0.0035%, 0.0030 % and 0.0025 %respectively have a greater zone size (mm) compare to thedifferent concentrations of Makabuhay (IIA-0.0045%, IIB-0.0040%, IIC- 0.0035%, IID- 0.0030 % and IIE- 0.0025 %).For the other pairs the mean differences are notsignificant.
62 CHAPTER V SUMMARY OF THE FINDINGS, CONCLUSION AND RECOMMENDATIONS This chapter brings together the results presentedwithin the previous chapter. Moreover, the importance ofthe study is completely stressed out.Summary of Findings The results and statistical analysis obtained aresummarized according to the order of the sub-problems ofthis study. The different results of the study undertakenare the following:1. The first sub-problem aimed to determine the mortalityrate of Nile Tilapia fingerlings after its exposure toTuba-tuba and Makabuhay extracts. The summary mortality rate of Nile Tilapia fingerlingsafter 96 hours of exposure to the different concentrationof Tuba-tuba were 5 tilapia fingerlings for 0.13%concentration and 4 tilapia fingerlings for 0.11%. On theother hand, the mortality rates for Makabuhay were 4tilapia fingerlings in 0.0045% concentration and 3 tilapiafingerlings in 0.0040% concentration. On the other hand,for the negative control the mortality rate of Tilapia
63fingerlings is 0. Moreover, the computed lethalConcentration 50 of Jatropha curcas (Tuba-Tuba) was 0.179and for Tinospora crispa (Makabuhay), the computed LC50 was0.0056 % which is lethal to the fingerlings.2. The second sub-problem aimed to determine the zone ofinhibition of Tuba-tuba and Makabuhay leaf extracts withdifferent concentrations and Tetracycline with the sameconcentrations as of both Tuba-tuba and Makabuhay. The zone of having the concentration of 0.13%, 0.11%,0.09%, 0.07% and 0.05% were the ff: 15.3 mm, 14.6 mm, 13mm, 0 mm, and 0 mm; while for tetracycline having the sameconcentrations, the zone of inhibitions were: 45.3 mm, 41.6mm, 39.3 mm, 38 mm and 35.3 mm. On the other hand, forMakabuhay leaf extract with the concentration of 0.0045%,0.0040%, 0.0035%, 0.0030% and 0.0025%, the recorded zone ofinhibitions were the ff: 18.6 mm, 16 mm, 14.3 mm, 13.6 mm,mm, and 12 mm; and for tetracycline with the sameconcentrations, the zone of inhibitions were the ff: 31.6mm, 28.6 mm, 27.6 mm, 27 mm, and 26.3 mm.3. The third sub-problem of this study aimed to determinethe differences among Tuba-tuba and Makabuhay leaf extractsand Tetracycline based on the zones of inhibition.
64 Jatropha curcas (Tuba-tuba) leaf extract andTetracycline with the same concentrations of 0.13%, 0.11%,0.09%, 0.07% and 0.05% has significant differences.Moreover, the leaf extract of Tinospora crispa (Makabuhay)and Tetracycline with the same concentrations of 0.0045%,0.0040%, 0.0035%, 0.0030% and 0.0025% also has significantdifferences.4. The fourth sub-problem aimed to determine the best leafextract among Tuba-tuba and Makabuhay leaf extracts. Based on the means of zone of inhibitions of Jatrophacurcas, it was observed that at 0.09% concentration,inhibition of growth of bacteria was manifested. On theother hand, the Tinospora crispa at the concentration of0.0025%, it was observed to inhibit growth of bacteria. However, comparing the two leaf extracts is notpossible, due to the fact that different concentrationswere used for each.Conclusions After 96-hours of exposure of Nile Tilapia fingerlingsinto the water containing leaf extracts of Tuba-tuba andMakabuhay, it was observed that there was low mortalityrate occurred. This led to the conclusion that all the
65concentrations used for each plant sample were considerednot lethal to Nile Tilapia Fingerlings in lined with thecomputed Lethal Concentration 50 of each sample. Measuring the zone of inhibition of leaf extracts ofTuba-tuba, Makabuhay, and Tetracycline showed thattetracycline has bigger measurement compare to the twoplant samples, respectively, despite that the sameconcentrations as of the plant samples were used fortetracycline. Comparing the zones of inhibition of Tuba-tuba,Makabuhay, and Tetracycline, the analysis showed that aresignificant differences between the leaf extracts of Tuba-tuba and Tetracycline, and between Makabuhay and ofTetracycline. It only implies that whether among the fiveconcentrations of Tuba-tuba, Makabuhay, and Tetracycline,will be used or be applied have different effects to theAeromonas sobria veronii. Moreover, the best leaf extract cannot be determinedsince different concentrations were used for each plantsamples. With these conclusions, the researchers are now ableto conclude for the main problem of this study.
66 The main problem of this study was to determine theantibacterial activity of Jatropha curcas (Tuba-tuba) andTinospora crispa (Makabuhay) against the fish pathogenAeromonas sobria. Tuba-tuba and Makabuhay leaf extract concentrationswere all not toxic to the Nile Tilapia Fingerling. Also, itwas shown that makabuhay and tuba-tuba has an inhibitoryeffect to the growth of Aeromonas sobria. However, incomparison with tetracycline, tetracycline is still moreeffective than the leaf extracts.Recommendations After obtaining the results discussed above thatanswer to the problems presented in the antibacterialactivity of Jatropha curcas and Tinospora crispa againstthe fish pathogen Aeromonas sobria the researchers havearrived at the following recommendations to further improvethe study:1. The study has focused only on the antibacterial activityof Jatropha curcas (Tuba-tuba) and Tinospora crispa(Makabuhay) against Aeromonas sobria. In lined with this,the researchers highly recommended the phytochemicalscreening of tuba-tuba and makabuhay for the identification
67of its specific active antimicrobial component.2. Future researchers are encourage to determine the effectof plant extracts, Jatropha curcas (Tuba-tuba) andTinospora crispa (Makabuhay) , in the metabolism and otherphysiological function of Nile Tilapia.3. Moreover, future researchers are recommended to use thesame plant sample but with different solvents other thanwater for extraction. This study may compare two or moresolvents, and identify what specific solvent is the bestfor Jatropha curcas and Tinospora crispa leaf extractions.4. Lastly, for future studies, it is recommended to useother parts of Jatropha curcas and Tinospora crispa andcompare the antibacterial activity of its different part.
68 BIBLIOGRAPHYBooksCamus, A.C., Durborow, R.M., Hemstreet, W.G., Thune, R.L. and Hawke, J.P, 1998, Aeromonas Bacterial Infections Motile Aeromonad Septicemia, Southern Regional aquaculture Center Publication, Alabama, pp. 1-2De Tevera, T.H. Pardo,2000, Plantas Medicinales de Filipinas, Jatropha Curcas L, Ayala foundation Inc., Makati Avenue, Ayala Triangle, Makati City, p.164-165De Tevera, T.H. Pardo,2000, Plantas Medicinales de Filipinas, Menispermaceae, Ayala foundation Inc., Makati Avenue, Ayala Triangle, Makati City, p.14El-Sayed, A.M., 2006, Tilapia Culture, Motile Aeromonas Septecemia, CABI Publishing, 875 Massachusetts Avenue 7th floor Cambridge, MA02139 USA, p.149Rodgers, C., and Furones, M.D., 2009, The use of Veterinary drugs and Vaccines in Mediterranean aquaculture, Antimicrobial agents in Aquaculture; Practice, needs, and issues, CIHEAM, Apartado 202-50080 Zaragoza,Spain, p. 47
69Romero, J., Feijoo. C. G., and Navarrete, P., April 11, 2012, Health and Environment in Aquaculture, Antibiotics in Aquaculture-Use, abuse and alternatives, In tech, p. 160JournalsAberoum, A., Jooyandeh, H., 2010, World Journal of Fish and Marine Sciences, A Review on Occurrence and Characterization of the Aeromonas Species from Marine Fishes, 6:p. 519Ahmed, W. A., and Salimon, J., 2009, European Journal of Scientific Research, Phorbol esters as toxic constituents of Tropical Jatropha curcas seed oil, 31:p.431Ashiru, Uaboi-Egbeni, Oguntowo, Idika, 2011, Pakistan Journal of Nutrition, Isolation and antibiotic profile of Aeromonas Species from Tilapia fish and Catfish, 10: pp.1- 2Badillo, L. J., 2006, Rev. Biol. Trop. Int. J. Trop. Biol., Age growth-models for Oreochromis aureus (Perciformes, Cichlidae) of the Infiernillo reservoir, Mexico and reproductive behaviour, 54: pp. 577-588
70Cipriano, R. C., 2001, Fish disease leaflet, Aeromonas hydrophila and Motile Aeromonad Septicemias of fish, 68: pp.1-2Das, K., Tiwari, R. K. S. and Shrivastava, D. K., 2010, Journal of Medicinal Plants Research, Techniques for evaluation of medicinal plant products as antimicrobial agent: Current methods and future trends, 4: p. 105Genghesh, K.S., Ahmed, S.F., El-Khalek, R.A., Al-Gendy, A., Klena, J., 2008, J Infect Developing Countries, Aeromonas in Developing countries, 2: pp. 82-83Haniffa, M.A and Kavitha K., 2012, Journal of Agricultural Technology, Antibacterial activity of medicinal herbs against the fish pathogen Aeromonas hydrophila, 8: p. 205Hossain, M., Univ. J.2001 Rajshahi Univ., 2008, Isolation of Pathogenic bacteria from the skin ulcerous symptomatic gourami (Colisa lalia) through 16rDNA analysis, 27: p.21Islam, A.K.M.A., Yaakob, Z., and Anuar, N., 2011, Scientific research and essays, Jatropha: A Multipurpose plant with considerable potential for tropics, 6: p.2600
71Janda, J. M., and Abbott, S. L., 2010, Clinical Microbiology Reviews, The Genus Aeromonas: Taxonomy, Pathogenicity, and Infection, 23: pp.44-45Michalova, E., Novotna, P., Schlegelova, J., 2004, Vet. Med. – Czech, Tetracyclines in veterinary medicine and bacterial resistance to them, 3: pp. 80-81Namuli, A., Abdullah, N., Sieo, C. C., Zuhainis, S. W., Oskoueian, E., 2011, Journal of Medicinal Plants Research, Phytochemical compounds and antibacterial activity of Jatropha curcas Linn. Extracts, 5: pp. 3984-3985Obi, R.K., Nwanebu, F.C, Naubuisi-Nnaji, U. U., Onuoha, L.N., and Chiegboka, N., 2011, Pharmacie Globale International Journal of Comprehensive Pharmacy, Ethanolic Extraction and Phytochemical Screening of two Negerian Herbs on Pathogens isolated from wound infections, 10: p.1Pandey, G., Sharma, M., and Mandloi, A. K., Plant Archives, Medicinal Plants useful in Fish Diseases, 12: pp.1-2
72Pompa, T., and Masser, M., 2012, Tilapia Life History and Biology, Southern Regional Aquaculture center. ,Rahman, T., Akanda, M. M. R., Rahman M. M. and Chowdhury, M. B. R., J. Bangladesh Agril. Univ., Evaluation of the efficacies of selected antibiotics and medicinal plants on common bacterial fish pathogens, 7:p.163Shayo S.D., Mwita C.J., and Hosea K., 2012, Open Access Scientific Reports, Ulcerative Aeromonas Infections in Tilapia (Cichlidae: Tilapiini) from Mtera Hydropower Dam, Tanzania, 1: pp. 1-2Published thesis/ unpublished thesisBecker, Klaus, Makkar, Harinder, P.S., Devappa, Rakshit, K. August 12, 2010, Jatropha toxicityCagauan, A. G., Galaites, M. J., and Fajardo, L.J., 2004, Evaluation of Botanical Piscisides on Nile Tilapia ( Oreochromis niloticus) and Mosquito fish Gambusia affinis BAIRD AND GIRARD, p. 183Cruz, M.C.S., 2000, New Antimicrobial Diterpenes from Tinospora rumphii
73Dabrowski, Konrad, 2012, Probiotics in Yellow Perch and Tilapia CultureIbrahem, M.D., Mostafa, M.M., Arab, R.M., Rezk, M.A., 2008, Prevalence of Aeromonas hydrophila infection in wild and cultured Tilapia nilotica (Oreochromis niloticus) in Egypt, p.2Electronic sourcesHasan, Jaful, March 2006, Aeromonas: Human Health Criteria Document,http://www.epa.gov/waterscience/criteria/humanhea lth/microbial/aeromonas 200603.pdfKhan, V., December 7, 2011, Online Guide to the Animals of Trinidad and Tobago-Nile Tilapia, http://sta.uwi.edu/fsa/lifesciences/documents/Oreochromis_ niloticus.pdfYanong, Roy P.E., January 2003, Use of Antibiotics in ornamental fish aquaculture, http://edis .ifas.ufl.eduFloyd, R. F., May 2009, Aeromonas infection, http://edis .ifas.ufl.ed