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THESIS
PASSIVE IMMUNITY LEVEL IN BROILER CHICKEN INFECTED WITH VARIOUS DOSE OF SPOROZOITE ON CAECAL COCCIDIOSIS
By: INANDA AYU SHABRINA 061111158
FACULTY OF VETERINARY MEDICINE UNIVERSITAS AIRLANGGA SURABAYA 2015
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S.
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THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Has been assessed in Result Seminar
Date: August 10, 2015
RESULT SEMINAR ASSESSMENT COMMITTEE
Head : Prof.Dr.Fedik A Rantam., drh.
Secretary : Prof.Dr.Lucia Tri Suwanti, drh., M.P.
Member : Rudy Sukamto Setiabudi, drh., M.Sc.
Supervisor : Prof. Mas’ud Hariadi, drh., M.Phil., Ph.D.
Co-Supervisor : Muchammad Yunus, drh, M.Kes., Ph.D
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S. iv
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PASSIVE IMUNITY LEVEL IN BROILER CHICKEN INFECTED WITH VARIOUS DOSE OF SPOROZOITE ON CAECAL COCCIDIOSIS
Inanda Ayu Shabrina
ABSTRACT
The aim of research is to know the passive immunity level in broiler chicken infected with various doses sporozoites of E. tenella on caeccal coccidiosis. The complete random design of research was used in this experiment. Forty of broiler divided into four treatments and each treatment composed ten replications. P0 was chicken group without serum administration then challenged with 8 x 104. P1, P2, P3 were chicken group with serum injection then challenged with 2 x104, 4x104, 8x104 respectively. Observation in this research were clinical symptoms, histopathological featuring (hemorrhage ,inflammation, villi rupture), and oocyst production. The data of clinical symptoms were analyzed descriptively. Oocyst production was analyzed using ANOVA and histopat scoring was analyzed using Kruskall-Wallis test. On sporozoite inoculation step after serum injection, level of clinical signs such as appetite, weakness, and diarrhea was negative on P1,P2,P3 groups. Passive immunity of P2 and P3 was relatively equal resistance. In P1 the resistance level given by passive immunity was the best compared to P2 and P3 so P1 was number of sporozoite dose in E.tenella which the most effectively affected the passive immunity level in broiler chickens infected with coccidiosis with 2x104 dose of E.tenella sporozoite. Key words: Eimeria tenella, sporozoites, passive immunity
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ACKNOWLEDGEMENT
My highest gratitude for Allah S.W.T. for blessing and mercy that this thesis
entitled PASSIVE IMMUNITY LEVEL IN BROILER CHICKEN INFECTED
WITH VARIOUS DOSE OF SPOROZOITE ON CAECAL COCIDIOSIS can be
completed.Shalawat and salam always dedicated to Prophet MuhammadS.A.W
who bring us from the darkness to the lightness. In arranging this thesis, I would
like to give my sincere gratitude to:
Prof. Hj. RomziahSidik, drh., Ph.D. as the dean, Dr. Anwar Ma’ruf, drh.,
M.Kes as the first vice dean, and Dr. Rr. Sri Pantja Madyawati, drh., M.Si. as the
head of academic division of the Faculty of Veterinary Medicine, Universitas
Airlangga.
My best supervisor committee, Prof. Mas’ud Hariadi, drh., M.Phil., Ph.D
andMuchammadYunus, drh, M.Kes., Ph.D. for the guidance, patience, advice, and
motivation all this time.The examiner committee, Prof.Dr.Fedik A Rantam.,
drh,Prof.Dr.Lucia Tri Suwanti, drh., M.P, Rudy Sukamto Setiabudi, drh., M.Sc.
for all the suggestion and correction.
The lecturers and entire staff of faculty member in the Faculty of Veterinary
Medicine, UniversitasAirlangga for their knowledge, help, information, and
encouragement.
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My beloved parents and my family for their endless love, pray, warmth,
protection, affection, motivation, and every little thing in between.
My research partner and helper ;TitahAprilia, Gita Fatmawati, IhsanAdi P,
thanks for accompany me. My best encourager, YeniPurbowati and Sunny
Krishna Smit, thanks for the support in my hardest time, for the help, concern,
happiness, companionship, and motivation. All members of IC : VanyaAgvira,
Benda Alifianti S, Ogen Sea, Gita F, Dewi M, Alif Aida S, Anisah, AriPrasetya,
Diana Feryka , DigkaBayu, Dinar Puspita S, Dona Astari N, Elsa Leonita,
Firdausy K M, Gavrila A, Gheby Indira G, Evant Grady, Hadi M H, HeningTyas,
IhsanAdi P, Karina R, Kartika P, Kemala S N, M Imam, Monica S, Muhammad H
F, RistaqulHusna B, Lilian, Tri Purna, Vidi P, Paviand ANDALAS, bunch of love
and thanks for those craziness and memories that have been created, I’m really
glad to be part of them.All my friends in IMAKAHI (IVSA INDONESIA),thanks
for those experiences I’ve ever had and stories that we shared. Also, thanks to
everyone that can’t be mention one by one that helpful during my study.
I, as the author, acknowledge that this writing is still lacking and far from
perfect. However, with a humble heart, I hope this research will be useful for the
advancement of science and may give a contribution to the veterinary medicine
field and all the people who needs it.
Surabaya, 20thAugust2015
Author
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CONTENTS
Page COVER ...... i
APPROVAL ...... ii
STATEMENT ...... iii
IDENTITY ...... iv
ABSTRACT ...... vi
ACKNOWLEDGEMENT ...... vii
CONTENTS ...... ix
LIST OF TABLES ...... xii
LIST OF FIGURES ...... xiii
LIST OF APPENDIX...... xv
ABBREVIATIONS AND SYMBOLIC MEANING ...... xvi
CHAPTER 1 INTRDUCTION ...... 1
1.1 Background ...... 1 1.2 The Formulation of Problem ...... 5 1.3 Theoritical Base ...... 5 1.4 The Aims of Research ...... 6 1.5 The Outcomes of The Research ...... 7 1.6 Hypothesis ...... 7
CHAPTER 2 LITERATURE REVIEW ...... 8
2.1 Review Of Parasites...... 8 2.1.1 EimeriatenellaCharacteristic ...... 8 2.1.2 Morphology of EimeriaTenella ...... 8 2.1.3 Life Cycle of EimeriaTenella ...... 10 2.1.4 Transmission of EimeriaTenella ...... 13 2.1.5 Pathogenesis of EimeriaTenella ...... 13 2.1.6 Clinical Signs ...... 13 2.1.7 Histopathology of Coccidiosis...... 14 2.2 Broiler Chicken...... 17 2.2.1 Chicken Classification ...... 17
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2.2.2 Broiler Chicken Potential ...... 18 2.3 Immunity InCoccidiosis ...... 19 2.3.1 Classification of Immunity ...... 19 2.3.2 Passive Immunity ...... 21 2.3.2.1 Naturally Acquired Active Immunity ...... 22 2.3.2.2 Artificially Acquired Passive Immunity ...... 22 2.4 Serum ...... 23
CHAPTER 3 MATERIAL AND METHODS ...... 25
3.1 Research Location and Date ...... 25 3.2 Project Research ...... 25 3.3 Material and Equipment of Research ...... 25 3.3.1 Experimental Animal ...... 25 3.3.2 Experimental Equipment ...... 25 3.3.3 Experimental Material ...... 25 3.4 Research Methods...... 26 3.4.1 Serum Collecting and Serum Storaging ...... 26 3.4.2 Serum Dosage Calculation ...... 26 3.4.3 Experimental Animal Adaptation ...... 26 3.4.4 Research Observation ...... 27 3.4.5 Observation Result ...... 27 3.4.5.1 Observation of Clinical Sign ...... 27 3.4.5.2 Observation of Oocyts Production and Histophatology...... 28 3.4.6 Research Variable ...... 29 3.4.7 Data Analysis ...... 30 3.5 Research Framework ...... 31
CHAPTER 4 RESEARCH RESULT ...... 32
4.1 Clinical Signs ...... 32 4.2 Oocyst Production ...... 32 4.3 Histopathologycal Changes ...... 33 4.3.1 Histopathologycal Changes Scoring ...... 33 4.3.2 Figure of Histopathologycal Changes Chicken Caecum .... 34
CHAPTER 5 DISCUSSION ...... 49
5.1 Clinical Symptomps ...... 49 5.2 Oocyst Production ...... 50 5.2.1 Daily Production of Oocyst ...... 50 5.3 Figure of Histopathological Changes Chicken Caecum ...... 50
CHAPTER 6 CONCLUSION ...... 53
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6.1 Conclusion ...... 53 6.2 Suggestion ...... 53
SUMMARY ...... 54
REFERENCES ...... 57
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LIST OF TABLES
Table Page 4.1 The Average Clinical Symptoms, Such as Fatigue, Decreasing Apetite,
Diarrhea, Bloody Diarrhea in Control Group and Treatment Groups
(P1,P2,P3) ...... 32
4.2 The Average Oocyst Production in Control Group and Treatment Groups
(P1,P2,P3) ...... 33
4.3 The Average Haemorrhage Scoring in Control Group and Treatment Groups
(P1,P2,P3) ...... 34
4.4 The Average Inflammation Scoring in Control Group and Treatment Groups
(P1,P2,P3)...... 34
4.5 The Average Vili Rupture Scoring in Control Group and Treatment Groups
(P1,P2,P3)...... 35
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LIST OF FIGURES
Figure Page 2.1 Structure of SporulatedEimeratenellaOocyst ...... 9
2.2 Eimeriatenella Life Cycle ...... 12
2.3 Histopathological Changes in the Chicken Caecum on Day 7 ...... 15
2.4 Histopathological of Normal Chicken Caecum ...... 15
2.5 Caecum Normal Chicken ...... 16
2.6 Chicken Caecum Which Infected Eimeriatenella ...... 16
2.7 Broiler Chicken ...... 17
2.8 Classification of Imunity ...... 21
3.1 Research Flow Chart ...... 31
4.1 Average Scoring of Histopathology Changements ...... 35
4.2 Inflammation in P0 Caused by InoculationsporozoitesE.tenella, magnification 100x ...... 37 4.3. Inflammation in P1 Caused by Inoculation sporozoitesE.tenella, magnification 100x ...... 38 4.4. Inflammation in P2 Caused by Inoculation sporozoitesE.tenella, magnification 100x ...... 39 4.5. Inflammation in P3 Caused by Inoculation sporozoitesE.tenella, magnification 100x ...... 40 4.6. Haemoraghe in P0 Caused by Inoculation sporozoitesE.tenella, magnification 100x ...... 41 4.7. Haemoraghe in P1 Caused by Inoculation sporozoitesE.tenella, magnification 100x ...... 42 4.8. Haemoraghe in P2 Caused by Inoculation sporozoitesE.tenella, magnification 100x ...... 43 4.9. Haemoraghe in P3 Caused by Inoculation sporozoitesE.tenella, magnification 100x ...... 44 4.10. Vili Rupture in P0 Caused by Inoculation sporozoitesE.tenella, magnification 100x ...... 45 4.11. Vili Rupture in P1 Caused by Inoculation sporozoitesE.tenella, magnification 100x ...... 46 4.12. Vili Rupture in P2 Caused by Inoculation sporozoitesE.tenella, magnification 100x ...... 47
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4.13. Vili Rupture in P3 Caused by Inoculation sporozoitesE.tenella, magnification 100x ...... 48
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LIST OF APPENDIX
Page
Appendix 1. Kruskallwallis test using SPSS for histopathological changes ... 61
Appendix 2. Kruskall Wallis test using SPSS for daily production oocyst ..... 73
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ABBREVIATIONSAND SYMBOLS MEANING
°C = Celcius Degree
µg = Microgram
ANOVA = Analysis OfVarians
cc = centimetre cubic
cm = centimeter
CP = Charoen Pokphand
CRD = Completely Randomized Design
dpi = day post infection
GALT = Gut Associated Lymphoid Tissue
Ig A = Immunoglobulin A
Ig G = Immunoglobulin G
IVIG = Intravenous Immunoglobulin
MAb = Monoclonal Antibodi
Mat Ab = Maternal Antibody
MSI = Macroscopic Lession Scoring
NaCl = NatriumChlorida
NK Cells = Natural Killer Cells
rpm = Rotation per Minute
SPs = Cysteine Protease
SPSS = Statistical Product and Service Solution
TCR = T-Cell Receptor
TEM = Transmission Electron Microscopy
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CHAPTER 1 INTRODUCTION
1.1 Background
Broiler chicken is a kind of livestock that many developed as a source of
animal protein supply and the most rapid growth, it because of broilers are the
result of cultivation that use the technology advanced so that has the properties
favorable economic (Syahbuddin, 2005).
Nowadays there are a lot of diseases that can attack chicken easily. One of
them is coccidiosis. Coccidia are common protozoa parasites, they are present in
all most type of chicken and Turkey flocks (Badran, 2006). Coccidiosis is one of
the most pathogenic intestinal diseases caused by different Eimeria species
belonging to phylum Apicomplexa (Nalbangtoglu et al., 2008). Heavy infection of
coccidia cause serious disease and will kill many chickens.
E.tenella is one Eimeria species that cause disease in chicken caecal
coccidiosis form that causes bloody diarrhea, weight loss, decreased egg
production and cause of death (Mc Dougald and Reid, 1991; Shierly et al., 1995;
Shierly, 1996). Coccidiosis cause the greatest loss compared with other chicken
diseases and estimated to reach 800 million dollars/year (Allen and Fatterer,
2002). In Indonesia, coccidiosis is one of chicken disease which almost always
appear in every chicken breeding period (Tabbu, 2006). Chicken of all ages can be
infected coccidiosis but 4-16 weeks old chickens are the most commonly easily to
get infected.(Badran, 2006).
Since every flocks is at risk from coccidiosis, all the 30 billion chickens
reared worldwide annually must be protected by prophylactic chemotherapy with
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specific drugs (most common) or by vaccination (increasing importance) (Shirley
and Johnson, 2001). On the other hand the development of anticoccidial resistance
has threatened the economic stability of the poultry industry (Tabbu,2006).
Although there has been little effort by the pharmaceutical industry to develop
new anticoccidials, the mountaining problem of drug resistance of Eimeria has
prompted major research efforts to seek alternative means of control through
vaccination. Some vaccines that have been tested are vaccines in the form of
attenuated whole oocyst (Shierly et al., 1995; Shierly, 1996). These vaccines can
only be used for chickens under 2 weeks old because chickens under 2 weeks old
can not digest oocyst (Yunus, 2007).
Eimeria infection in chickens primarily confined to the intestinal track and
the gut associated lymphoid tissue (GALT) which serves as the first line immune
defense against colonization by this organism (Lillehot et al., 2000). GALT serves
three functions in host defense against Eimeria processing and presentation of
antigens, production of intestinal antibodies (primarily secretary IgA), and
activation of cell-mediated immunity (Ganguly and Waldman, 1980; Brandtzaeg
et al., 1989; Neutra et a.l, 1996 and Yun et al., 2000). Studies revealed three
phenomena responsible for immunity against Eimeria infections. First, the actual
passage and presence of parasites in the lamina propria to induce immunity.
Second, the sporozoite seems to be the most important parasite stage for immunity
and third cytotoxic, T cells are necessary to inhibit parasites (Jeurissen et al.,
1986).
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Vaccines typically need time (weeks or months) to produce protective
immunity in an individual and may require several doses over a certain period of
time to achieve optimum protection. A different type of immunity, called passive
immunity. When the antibodies are introduced into the animal’s body, the
“loaned” antibodies help prevent or fight certain infectious diseases. There are
two types of passive immunity which are natural passive immunity and artificial
passive immunity. Natural passive immunity is the protection provided by the
mother, artificial passive immunity when antibodies are given as a medication to a
non immune individual.
Immunity elicited in one animal can be transferred to another animal by
injecting the recipient animal with serum from the immunited animal. The
protection offered by passive immunization is short lived, usually lasting only a
few weeks or months. On the other hand it helps protect right away cause
producing an immune response within hours (2-3 hours) or days, faster than active
vaccine. Such immunity is species specific and possibly directed against the
sporozoite stage. It has been shown recently that extracts from the various doses
of sporozoites of E. tenella containing no viable parasites can be used to
successfully vaccinate against the infection. The effector mechanism of protective
immunity induced by live infection or by extract vaccine. By given the role of
serum antibody in protective immunity.
Serum is the liquid fraction of clotted blood. It is depleted of cells, fibrin, and
clotting factors. Serum differs from plasma in that anti coagulant is never added to
the blood after collection from the animal. Serum is prepared by centrifuging until
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the clot and remaining blood cells are separated from the liquid phase. The serum
removed and stored frozen pending futher processing. Serum is very complex
supplement containing mostly proteins, but also growth factors, hormones, amino
acids, glucose, trypsin inhibitors, and lipids. Animal serum is an excellent source
of nutrients for cells in culture because it contains proteins, lipids, salts, vitamins,
minerals, amino acids and other components necessary for growth. When stored
and handled correctly, the performance characteristics of serum can be maintained
for many years.
Improperly storing and thawing serum products can decrease not only the
immediate and long-term stability, but also their effectiveness. Growth promotion
data demonstrate that serum maintains its growth characteristics throughout its
shelf life if stored correctly.To effectively preserve the integrity of animal serum, it
should be stored frozen and protected from light. The recommended storage
temperature is -10 to -40 °C. At temperatures below -40 °C, the bottles may become
brittle resulting in an increased risk of breakage. Multiple thaw/freeze cycles should
be avoided as they will hasten the degradation of serum nutrients and can induce the
formation of insoluble precipitates. Furthermore, serum should never be stored in
“frost-free” freezers. These appliances occasionally warm themselves to avoid
internal ice deposits and are detrimental to the clarity and stability of frozen serum
products.
Based on a reasons above, the passion study is to do a further research in
vaccination for coccidiosis case in broiler chicken. Especially to know passive
immunity particulary rise up passive immunity with various dose of sporozoite
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on coccidiosis infection. Some indicators of the presence of protective immunity
in coccidiosis can be represented in oocyst production, pathological anatomy
changes, and clinical changes.
1.2. The Formulation of Problem
1. Is there immunity in coccidiosis infection broiler chickens after the
administration of serum of infected chicken by E. tenella sporozoites ?
2. What is the optimal dose of E. tenella that can be given to form immunity
after the administration of passive immunity?
1.3. Theoretical base
The obligate intracellular apicomplexan parasites cause devastating
diseases in humans and domestic animals. Well-known members of this phylum
are Plasmodium falciparum, Toxoplasma gondii, Cryptosporidium parvum,
Theileria annulata and E. tenella (Mehlhorn, 2008; Morrison, 2009). The poultry
intestinal disease known as coccidiosis is caused by Eimeria spp. such as Eimeria
acervulina, Eimeria necatrix, E. tenella and Eimeria mivati. The most pathogenic
species, E. tenella, provokes a haemorrhagic diarrhea in young chickens, leading
to a loss of weight and appetite, resorption problems, bacterial secondary
infections and often also to the bird’s death (Chauhan and Roy, 2007; Mehlhorn,
2008; Shirley et al., 2007).
It has long been known that infection with any of the species of Eimeria
can induce a potent protective immune response in the host that is exquisitely
specific to each species of parasite (Beach and Corl., 1925; Edger, 1958). It was
observed that if fowl immunized by E.tenella when given an additional infection
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with E.necatrix, produced precipitates in the serum that reacted with antigents of
both the species (Rose and Long, 1962). Solid immunity to any species occurred
after infections but some species like E.tenella and E.maxima are so highly
immunogenic that an intake to of only few oocysts can induce almost complete
immunity to homologous challenge (Devies et al., 1963). The authors used
irradiated oocysts such thas sporozoite invasion of the intestinal epithelium was
unaffected but that subsequent merogonic development was inhibited.studies
supporting the notions that the asexual stage appeared to be more functionally
immunogenic than the sexual stages or sporozoite stage indicated that even among
the asexual stages, some are more effective than others at inducing (McDonald et
al., 1988; Tomley, 1994).
Various efforts have been done to deal with this disease but nothing have
fully worked out until now. Preventive action using anti parasite causes resistance
and left residual substance, both in meat and eggs. Preventive actions by giving
vaccine or serum for chickens is now being developed. Vaccine through passive
immunity provides immediate protection. This study aims to determine the
number of sporozoites dose in E. tenella which affect the passive immunity level
of broiler chickens most effectively judged by the clinical symptoms, oocyst
production, and histopathological changes view in broiler chickens.
1.4. The Aims of Research
1. To investigate how’s passive immunity in broiler chickens after the
administration of serum of infected chicken by various doses of E. tenella
sporozoites
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2. To investigate the number of sporozoites doses in E. tenella which the most
effective affect on passive immunity level for broiler chicken infected
coccidiosis.
1.5. The Outcomes of the Research
1. The research can be used as an information that has ability to induce
sporozoite in passive immunity level
2. The research can be used as a reference to increase development of poultry
farm in Indonesia, especially as prevention of coccidiosis in broiler chicken
1.6. Hypothesis
1. Administration serum of infected chicken by E. tenella sporozoites provided
the formation of passive immunity in coccidiosis infection broiler chicken.
2. There is the optimal dose of E. tenella that can be given to form immunity
after the administration of passive immunity.
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CHAPTER 2 LITERATURE REVIEW
2.1. Review of parasites
2.1.1. Eimeria tenella Characteristic
E. tenella is monogenetic. Various stages of its complicated life cycle may
conveniently be described under two phases, asexual cycle or schizogony and
sexual cycle involving gametogony. It is one of seven protozoan parasites that
cause avian Coccidiosis in poultry.
The Taxonomy of Eimeria tenella (Soulsby, 1985)
Kingdom :Protista
Phylum :Apicomplexa
Class :Conoidasida
Order :Eucoccidioria
Sub Order :Eimeriina
Family :Eimeriidae
Genus :Eimeria
Species :Eimeria tenella
2.1.2. Morphology of Eimeria tenella
The invasion and replication of E. tenella in the chicken intestine is
responsible for avian coccidiosis, a disease that has major economic impacts on
poultry industries worldwide (Anaïs Rieux et al., 2012). Between the seven
species of Eimeria commonly detected in infected chickens, E. tenella is one of
the most virulent and the only one for which the genome has been sequence. E.
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tenella is transmitted to naïve animals via shed unsporulated oocysts that need
contact with air and humidity to form the infectious sporulated oocysts, which
contain the first invasive form of the parasite, the sporozoite. Cysteine proteases
(CPs) are major virulence factors expressed by protozoa. E. tenella oocyst has
avoid width shape, with lengths ranging from 14.2 to 31.2 microns and a width of
9.5 to 24.8 microns with two layered wall consisting of an outer layer and inner
layer. Wall of oocyst is smooth and does not have microphily. Oocyst in optimal
conditions will mature and into the infective oocyst (sporulated). The time needed
of E. tenella to sporulate in 18 hours to two days. In the sporulated oocyst there
are 4 of each sporocyst - each containing 2 sporozoites. E. tenella to be sporulated
take up to 18 hours at a temperature of 29oC, 21 hours is 26 - 28ºC, 24 hours is
20-24ºC and 24-28 hours in room temperature and can’t be sporulated at
temperatures below 8ºC. (Soulsby, 1986).
Figure 2.1. (a) structure of sporulated Eimeria oocyst ; (b) sporulated Isospora oocsyst (Levine,1971)
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2.1.3. Life cycle of Eimeria tenella
E. tenella is one of protozoa which cause coccidiosis or bloody diarrhea in
chicken. It is a major cause of disease and may be associated with a high rate of
mortality, particularly among young chickens. The life cycle of coccidian parasites
involves asexual and sexual development in the gut with location depending on the
specific species. The result of the sexual development is the production of resistant
cysts (oocysts) that are secreted with the feces. In the external environment the
oocysts under development (sporulation) to form infectious oocysts, which have a
characteristic appearance.
As for other Coccidia, the complex life cycle of E. tenella is divided into
an intestinal and an environmental stage. The intestinal stage involves the
invasion and replication of parasites within epithelial cells of the chicken
intestine, followed by production of male and female gametes, fertilisation and
formation of unsporulated oocysts, which are released into the environment. The
environmental stage involves maturation, also called sporulation, of unsporulated
oocysts into infectious sporulated oocysts. Each sporulated oocyst contains eight
haploid sporozoites, the first invasive form of the parasite.
The oocysts contain four sporocysts, each containing two sporozoites (the
infectious stage). These oocysts transmit the infection to new susceptible hosts
when ingested. The oocyst is a unique structure in its ability to survive in the
external environment for extended periods, making control of infection very
difficult. The oocyst is protected by a wall that is resistant to chemical and physical
insult. Oocysts are sensitive to heat and desiccation.
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Zymes of the gut it undergoes excystation to release the eight infectious
sporozoites. The sporozoites are able to invade the epithelial cells of the caecum
and undergo asexual multiplication to produce first generation schizonts. The
merozoites produced then invade new epithelial cells to give rise to second
generation schizonts. Large groups of second-generation schizonts are located
within the mucosa and cause ulceration of the mucosal surface. In a section through
the caecum shows a large number schizonts (stained brown) that are causing
disruption of the mucosa. At higher power it can be seen that the mature schizonts
contain large numbers of merozoites . In a detailed image it is possible to identify
an immature schizonts adjacent to mature schizonts. When examined by scanning
electron microscopy the surface features of an early schizont show the initiation of
the formation merozoites as conical protrusions from the surface. This process
results in the formation of vacuoles containing the fully formed cigar shaped
merozoites. These merozoites escape from the host cell and are able to move using
a flexing motion . The merozoites when examined by transmission electron
microscopy (TEM) contain a single nucleus and numerous organelles at the front
(apical) end that are termed the conoid, rhoptries and micronemes . Specific
molecules within these or ganelles are involved in the recognition, adhesion and
invasion of new host cells.
TEM it can be seen that microgametes form by protruding from the surface of
the microgametocyte. At this stage the two flagella and nucleus can be identified.
At the mature stage numerous coiled microgametes can be observed. This budding
of the microgametes into the vacuole can be seen by TEM. In the case of the of the
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macrogamete, the surface appearances are relatively smooth. However when the
interior is examined it is remarkably complex. The developing macrogamete is
required to synthesis all the components needed for oocyst formation and survival.
By immunostaining, it can be seen that a number of granules are formed that will be
responsible for oocyst wall formation and that there is carbohydrate storage in the
form of numerous polysaccharide granules. The different types of granules can be
more easily identified by TEM. The macrogamete develop into the oocyst at the
time of fertilisation by the microgamete. The interior of the developing oocyst is
enclosed by a dense wall, which is formed with the accompanying loss of the
electron dense cytoplasmic granules present in the macrogamete. Developing
oocysts with associated microgametes have been observed. A typical feature is the
slight shrinkage resulting in the ridged appearance of then developing oocyst. This
is related to the resistant nature of the wall which protects the oocyst in the external
environment. The oocysts are then excreted with the faeces (Fergusson, 2003).
Figure 2.2. Eimeria tenella life cycle (Barta, 2001)
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2.1.4. Transmission of Eimeria tenella
Birds infected with coccidia may shed oocysts in their feces for days or
weeks. Susceptible birds in the same flock ingest the infective or sporulated
oocysts in the litter, soil, feed or water and become infected. Infected feces or
litter on boots, clothing, equipment, darkling beetles or in dust can then spread the
disease to other houses or farms. Outbreaks occur when susceptible birds ingest
massive numbers of sporulated oocysts. The best conditions for oocyst sporulation
occurs in wet litter with warm temperatures. Oocysts can survive up to 4 years in
the environment if conditions are right (Helm, 1999).
2.1.5. Pathogenesis of Eimeria tenella
E. tenella is one of the most pathogenic Eimeria species to chickens
(Tabbu, 2006). This disease primarily affects young chickens and cause high
mortality located in the epithelial cells of the caecum. While chronic infection in
adult chickens can lead to weight loss (Soulsby, 1986). The most pathogenic
Stadium on E. tenella is the second generation schizont, which are large and can
contain hundreds of merozoites. In general, the death occurred on day 5 to 6
post-infection. In acute infections, death can occur within a few hours after the
initial clinical symptoms occurred. In a number of cases of deaths caused by the
rupture in the cecum (Tabbu, 2006).
2.1.6. Clinical Signs
Clinical signs of coccidiosis can range from none to bloody droppings,
watery diarrhea (flushing), weight loss, paleness, sick bird appearance (ruffled
feathers, huddling, depression). Affected birds do not eat and will sometimes
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march from feed pan to feed pan, vocalizing a high pitched cry. Mortality can
range from mild to severe, depending on the species of coccidia involved. All
ages of poultry are susceptible to infection, but the disease usually resolves itself
around 6 – 8 weeks of age. The birds are most sensitive between 3 – 5 weeks of
age, when the coccidia oocysts are the most numerous in faeces or litter. Many
times the disease is subclinical and the flock may only show poor weight gains or
feed conversions at the end of grow-out (Helm, 1999).
2.1.7. Histopathology of Coccidiosis
(Suprihati et a.l, 2000) cited by Rahmawati (2013) states that the results of
microscopic examination of the cecum in chickens infected with 5000 oocysts
visible cell necrosis, degeneration, thickening of the muscular layer, infiltration of
inflammatory cells, the presence of parasites were surrounded by inflammatory
cells and hemorrhage. Caecum histopathology chickens infected with oocysts
showed damage to the caecum. Mufasirin (2012) mention that E. tenella infection
with ptecial until bleeding was found early in the disease. Enlarged cecum and
contains blood clots. In the case of chronic intestinal wall thickening and fibrosis
experience.
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Figure 2.3. Histopathological changes in the chicken cecum necropsy on day 7 after infected with 5000 oocysts Eimeria tenella. 20x10 magnification (Rohayati, 2011). Notes: a. Extravascular erythrocytes, b. Schizont, c. Inflammatory cells, d. Zygote
Figure 2.4. Histopathology of normal chicken caecum. 10x10 magnification (Rohayati, 2011). Notes: a. Tunica mucosa, b. Tunica submucosa, c. Tunica muscularis, d. The tunica serosa.
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Figure 2.5. Caecum normal chicken ( Cahyaningsih, 2003)
Figure 2.6. Chicken caecum which infected Eimeria tenella with a lesion score of +2 (A), cecum Eimeria tenella infected chickens with lesion score of +4 (b). (Cahyaningsih, 2013).
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2.2. Broiler Chicken
2.2.1. Chicken Classification
The Taxonomy of chicken
Kingdom : Animalia
Phylum : Chordata
Class : Aves
Order : Galliformes
Family : Phasianidae
Sub Family : Phasianidae
Genus : Gallus
Species :G. gallus
Sub Species G.g.Domesticus
Figure 2.7. Broiler Chicken (Allaboutfeed, 2011)
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2.2.2. Broiler Chicken Potential
Broiler is a term to call chicken strain cultured technology that has
characteristics economical, which is has rapid growth as producer meat, efficient
feed conversion, ready to cut in a relatively young age, and also produce quality
soft fiber meat (Murtidjo, 1987). Broiler chicken is a kind of livestock that many
developed as a source of animal protein supply and the most rapid growth, it
because of broilers are the result of cultivation that use the technology advanced
so that has the properties favorable economic (Syahbuddin, 2005).
The government has promoted the cross bred-chicken broiler industry through
government regulations. Since 1980, the government of Indonesia has limited the
scale of output. Government regulation (Keppres) No 50/81 stated that no farmer
can rear more than 5,000 chicken layers, or 750 chicken broilers per period of
production, and no large-scale production is permitted (Yusdja and Pasandaran,
1999). Keppres No. 22/70 permits rearing as many as 15,000 chicken layers and
2,500 chicken broilers per production period. The regulations permit large-scale
producers to operate with either domestic or foreign investments provided
production is for export and is done in partnership with small-scale firms (Saptana
and Suhartini, 1995; Syam, 1998; and Yusdja and Pasandaran, 1999). In
partnerships, the large firms supply working capital and technology and sell the
product of the small producers (Yusdja and Pasandaran, 1999).
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2.3. Immunity in Coccidiosis
2.3.1. Classification of Immunity
Immunity is the state of protection against infectious disease conferred
either through an immune response generated by immunization or previous
infection or by other non-immunological factors. In biology, immunity is the state
of having sufficient biological defences to avoid infection, disease, or other
unwanted biological invasion. It is the capability of the body to resist harmful
microbes from entering it. Immunity involves both specific and non-specific
components. The non-specific components act either as barriers or as eliminators
of wide range of pathogens irrespective of antigenic specificity. Other components
of the immune system adapt themselves to each new disease encountered and are
able to generate pathogen-specific immunity.
Innate immunity, or non specific immunity is the natural resistances with
which a person is born. It provides resistances through several physical, chemical
and cellular approaches. Microbes first encounter the epithelial layers, physical
barriers that line skin and mucous membranes. Subsequent general defences
include secreted chemical signals (cytokines), antimicrobial substances, fever, and
phagocytic activity associated with the inflammatory responses. The phagocytes
express cell surface receptors that can bind and respond to common molecular
patterns expressed on the surface of invading microbes. Based on these
approaches, non specific immunity can prevent the colonization, entry and spread
of microbes.
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Adaptive immunity is often sub-divided into two major types depending on
how the immunity was introduced. Naturally acquired immunity occurs through
contact with a disease causing agent, when the contact was not deliberate, whereas
artificially acquired immunity develops only through deliberate actions such as
vaccination. Both naturally and artificially acquired immunity can be further
subdivided depending on whether immunity is induced in the host or passively
transferred from an immune host. Passive immunity is acquired through transfer
of antibodies or activated T-cells from an immune host, and is short
lived—usually lasting only a few months—whereas active immunity is induced in
the host itself by antigen and lasts much longer, sometimes lifelong. The diagram
below summarizes these divisions of immunity.
A further subdivision of adaptive immunity is characterized by the cells
involved which is humoral immunity, whereas the protection provided by cell
mediated immunity involves T-lymphocytes alone. Humoral immunity is active
when the organism generates its own antibodies, and passive when antibodies are
transferred between individuals. Similarly, cell mediated immunity is active when
the organisms own T-cells are stimulated and passive when T cells come from
another organism. T cells or T lymphocytes are a type of lymphocyte (itself a type
of white blood cell) that play a central role in cell-mediated immunity. They can
be distinguished from other lymphocytes, such as B cells and natural killer cells
(NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. They
are called T cells because they mature in the thymus (although some also mature
in the tonsils)
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. Humoral immunity, also called the antibody-mediated beta cullularis immune
system, is the aspect of immunity that is mediated by macromolecules (as opposed
to cell-mediated immunity) found in extracellular fluids such as secreted
antibodies, complement proteins and certain antimicrobial peptides. Humoral
immunity is so named because it involves substances found in the humours, or
body fluids.,
Figure 2.8. Classification of immunity
2.3.2. Passive Immunity
Passive immunity refers to the process of providing IgG antibodies to
protect against infection. It gives immediate, but short-lived protection—several
weeks to 3 or 4 months at most. Passive immunity is usually classified as natural
or acquired. The transfer of maternal tetanus antibody (mainly IgG) across the
placenta provides natural passive immunity for the newborn baby for several
weeks/months until such antibody is degraded and lost. In contrast, acquired
passive immunity refers to the process of obtaining serum from immune
individuals, pooling this, concentrating the immunoglobulin fraction and then
injecting it to protect a susceptible person. Passive immunity can occur naturally,
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when maternal antibodies are transferred to the fetus through the placenta, and can
also be induced artificially, when high levels of human (or horse) antibodies
specific for a pathogen or toxin are transferred to non-immune individuals.
Passive immunization is not used when there is a high risk of infection and
insufficient time for the body to develop its own immune response, or to reduce
the symptoms of on going or immunosuppressive diseases. Passive immunity
provides immediate protection, but the body does not develop memory, therefore
the patient is at risk of being infected by the same pathogen later.
2.3.2.1. Naturally Acquired Passive Immunity
Maternal passive immunity is a type of naturally acquired passive
immunity, and refers to antibody-mediated immunity conveyed to a fetus by its
mother during pregnancy. Maternal antibodies (MatAb) are passed through the
placenta to the fetus by an FcRn receptor on placental cells. This occurs around
the third month of gestation. IgG is the only antibody isotype that can pass
through the placenta. Passive immunity is also provided through the transfer of
IgA antibodies found in breast milk that are transferred to the gut of the infant,
protecting against bacterial infections, until the newborn can synthesize its own
antibodies.
2.3.2.2. Artificially Acquired Passive Immunity
Artificially acquired passive immunity is a short-term immunization induced
by the transfer of antibodies, which can be administered in several forms; as
human or animal blood plasma, as pooled human immunoglobulin for intravenous
(IVIG) or intramuscular (IG) use, and in the form of monoclonal antibodies
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(MAb). Passive transfer is used prophylactically in the case of immunodeficiency
diseases, such as hypogammaglobulinemia. It is also used in the treatment of
several types of acute infection, and to treat poisoning. Immunity derived from
passive immunization lasts for only a short period of time, and there is also a
potential risk for hypersensitivity reactions, and serum sickness, especially from
gamma globulin of non-human origin.
The artificial induction of passive immunity has been used for over a century
to treat infectious disease, and prior to the advent of antibiotics, was often the only
specific treatment for certain infections. Immunoglobulin therapy continued to be
a first line therapy in the treatment of severe respiratory diseases until the 1930s,
even after sulfonamide lot antibiotics were introduced. Passive or "adoptive
transfer" of cell-mediated immunity, is conferred by the transfer of "sensitized" or
activated T-cells from one individual into another. It is rarely used in humans
because it requires histocompatible (matched) donors, which are often difficult to
find. In unmatched donors this type of transfer carries severe risks of graft versus
host disease. It has, however, been used to treat certain diseases including some
types of cancer and immunodeficiency. This type of transfer differs from a bone
marrow transplant, in which (undifferentiated) hematopoietic stem cells are
transferred.
2.4 Serum
serum is the liquid fraction of clotted blood. it is depleted of cells, fibrin, and
clotting factors. serum differs from plasma in that anti coagulant is never added to
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the blood after collection from the animal. Serum is prepared by centrifuging
untul the clot and remaining blood cells are separated from the lliquid phase.
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CHAPTER 3 MATERIAL AND METHODS
3.1. Research Location and Date
The experiment was performed at laboratory Department of parasitology and
research cage Faculty of Veterinary Medicine Airlangga University Surabaya,
Indonesia. The duration of the experiment was two months, in April till May
2015.
3.2. Project Research
This research was an experimental laboratory. The experimental design use a
completely randomized design (CRD) with the assumption of stable conditions,
animal, and treatment in a uniform condition.
3.3. Material and Equipment of Research
3.3.1. Experimental Animal
Experimental animals that used in this study were 40 strains of broiler
chickens two weeks old production Charoen Pokphand CP 707.
3.3.2 Experimental Material
The materials needed for this study were chicken feed without
coccidiostat, water, serum, various dose of sporozoites, saturated salt solution,
aquadest, alcohol, and aqua proinjection.
3.3.3 Experimental Equipment
The equipments that used for this study are battery cages. Another tools
used are microscope, improve neubauer, object glass, surgical scissors, tweezers,
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scalpel, centrifuge tube, centrifuge, measuring cups, syringes, microtome, pasteur
pipette, becker glass, filter, and gauze.
3.4. Research Methods
3.4.1. Serum collecting and serum storaging
Serum collecting was obtained from previous research (Riadiani, 2014).
Serum obtained from chicken blood that has been administered E. tenella
sporozoites then storaging serum in temperatur-10 to -40 °C. To effectively
preserve the integrity of animal serum, it should be stored frozen and protected
from light. At temperatures below -40 °C, the bottles may become brittle resulting
in an increased risk of breakage.
3.4.2. Serum dosage calculation
Immunity elicited in one animal can be transferred to another animal by
injection the serum (Passive Immunity). Serum which has been storaged in
temperatur -10 to -40°C was examined by nano spectofotometer method to find out
which the best serum dosage from all the group treatments (P1,P2,P3) in the
previous research. The serum which was administered to chicken were the whole
serum with the dose of 500 µg.
3.4.3. Experimental animal adaptation
Two weeks old 40 broiler chickens adapted in battery cages for one week
before the treatment. Two weeks old chickens are kept in battery cages. Given
feed which does not contain a coccidiostat and drink ad libitum.
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3.4.4. Research Observation
The research was conducted in separated groups as follows :
P0 : Native chickens were administered intravena with 0,5 cc aqua
proinjection then after 3 hours challenged with 8x104 sporozoites of E.
tenella which was administered peroral
P1 : Native chickens were administered intravena with 0,5 cc serum from
8x104 doses inoculated E. tenella sporozoites then after 3 hours
challenged with 2x104 sporozoites of E. tenella which was administered
peroral.
P2 : Native chickens were administered intravena with 0,5 cc serum from
8x104 doses inoculated E. tenella sporozoites then after 3 hours
challenged with 4x104 sporozites of E. tenella which was administered
peroral.
P3 : Native chickens were administered intravena with 0,5 cc serum from
8x104 doses inoculated E. tenella sporozoites then after 3 hours
challenged with 8x104 sporozoites of E. tenella which was administered
peroral.
3.4.5. Observation Result
3.4.5.1. Observation of clinical signs
Observation of clinical signs seen by chicken which was infected E. tenella
have changenment in chicken body condition such as : limp, decreasing apetite, and
bloddy diarrhea. Great blood diarhea happened in day-5 and day-6. If after
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innoculated sporozoites of E.tenella then challenged by administered serum there
was nothing change in chicken, it’s mean that the chicken already has immune from
the serum in the body.
3.4.5.2. Observation of oocyts production and histopathology
Observation of oocysts production was conducted by collecting the feses
of chickens. Take 2 gram of faeces each chicken and pour with 58 cc of saturated
salt till homogen then put inside Mc Master chamber and the oocysts can be seen by
microscop. The total number of oocysts were found and multiplied 100 then
multiplied again by the weight of faeces (Yunus,2007).
Microscopic observation was done based on histopathology chagement
in caecum and scored by Goodwin scoring method (2012) and modified by
(Suprihati et al., 2000) cited by (Ni’,matul, 2006), histopathological changes in
preparations was assessed with 5 scores seen in the visual field scored based on
inflammation, hemorrhage and parasite stage. According to Godwin et al, the
result of histopathological changes observation based on sum from A and B which
is A for distibrution the existance of E. tenella parasite in mucous caecum and B
for the damage level of villi in caecum in histopathological changes as follows :
Score 0 : if in 10 fields view appear the vili rupture level 0% cause of
pasites infection
Score 1 : if in 10 fields view appear the vili rupture level <25% cause of
parasites infection
Score 2 : if in 10 fields view appear the vili rupture level 25% - 50%
cause of parasites infection
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Score 3 : if in 10 fields view appear the vili rupture level; 51% - 75%
cause of parasites infection
Score 4 : if in 10 fields view appear the vili rupture level >75% cause of
parasites infection
Microscopic examination also modified conducted by histopahological
changes in preparations was assessed with five scores seen in the visual field and
the field of view is given a score according to (Suprihati et al., 2000) cited by
Ni’matul (2006) as follows :
Score 1 : if in ¼ field view appear inflammation, haemorrage, and there
is parasites stage.
Score 2 : if in ½ field view appear inflammation, haemorrage, and there
is parasites stage.
Score 3 : if in ¾ field view appear inflammation, haemorrage, and there
is parasites stage.
Score 4 : : if in 1 field view appear inflammation, haemorrage, and there
is parasites stage.
3.4.6. Research Variable
Variable of this research were defined into independent variables,
dependent variables and controlled variables.
Independent variable :Serum from 8x104 doses sporozoite E. tenella and
various doses of sporozoites E. tenella (2x104,4x104 and 8x104).
Dependent variables :Oocyst production in chickens,clinical signs and
histopathological changes.
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Controlled variables : Broiler chicken type, broiler chicken size, broiler
chicken body weight,cage, water and food.
3.4.7. Data Analysis
Research design used completely randomized design (CRD) with 4
treatments and 10 repititons for each treatment, based on t(n-1)≥5 function
(Kusriningrum, 2008). Data from histopathology caunted by Kruskall-Wallis.
oocysts production in faeces caunted using ANOVA to see the effect of
administrated serum in chicken immunity level. If there is significant effect in the
calculation then will be continued by Tukey Test or BNJ (Beda Nyata Jujur). Data
analyse were done by using software SPSS 21 for Windows.
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3.5. Research framework
Serum isolation from 40 chickens 2 weeks old the previous research
Adaptation for a week
Serum calculation Intravenal serum injection 8x104 doses using Nano inoculated Eimeria tenella sporozoites spectofotometer except for P0 (500 µg)
after 3 hours challenged with various serum storage dose of sporozoite E.tenella
P0 P1 P2 P3 Administe Administe Administ Administe red red ered red peroral peroral peroral peroral 8x104 2x104 4 4 4x10 8x10 sporozoite sporozoite sporozoit sporozoite E. tenella E. tenella E. E. tenella e tenella
4 dpi 5 chickens eutanized each treatment to see histopathology in caecum
4dpi Observation of oocyst E.tenella production and clinical signs for 7 days
Data analysis
Figure 3.1. Research flow chart
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CHAPTER 4 RESEARCH RESULT
4.1. Clinical Signs
Clinical symptomps shown on the control group (P0) for inoculation using
E.tenella sporozoites shown a very clear symptomps such as fatigue, decreasing
appetite, diarrhea and bloody diarrhea on the 5-6th day post inoculation and
dehydration. While the clinical symptomps in P1,P2,P3 which were given serum
first and then incoculated with Eimeria tenella sporozoites didn’t show any
significant symptomps.
Table 4.1. The average clinical symptomps such as fatigue, decreasing apetite, diarrhea, bloody diarrhea, dehydration in control group and treatment groups (P1,P2,P3)
Treatment Clinical Symptomps Fatigue Decreasing Diarrhea Bloody Appetite Diarrhea b P0 + + + + P1a - - - - a P2 - - - - P3a - - - -
4.2. Oocyst Production
4.2.1 Daily production of oocyst
Based on data from this research, daily production of oocyst between P0
and group treatments (P1,P2,P3) has a significant difference (P<5) is shown in
table 4.2. The table shows the oocyst production on each treatment per day after
serum administration and inoculation using various dose of sporozoites.
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Table 4.2 Daily ooscyst production pattern between control group which was not administrated serum intravenous injection then challenged with 80000 sporozoites and treatment groups (P1,P2,P3) which was administrated serum intravenous injection then challenged with various doses of sporozoites (20000,40000,80000).
Treat Day post inoculation ment Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 P0 32140b± 15660b± 16940b± 24160b± 37040b± 31440b± 352001 b± 2940.7 96756.4 44461.2 88976.4 126264.8 1384.0 551.8 P1 0a±0000. 2200a±4 0a±0000. 0a±0000. 0a±0000. 1080a±2 4200a±93 0 919.3 0 0 0 414.9 91.4 P2 0a±0000. 3600a±8 0a±0000. 7000a±9 4000a±8 2600a±5 4000a±89 0 049.8 0 746.7 944.2 813.7 44.2 P3 0a±0000. 0a±0000. 9800a±1 0a±0000. 5960a±7 4400a±9 4200a±65 0 0 342.8 0 956.1 838.6 72.6
4.3 Histopathologycal Changes
4.3.1 Histopathologycal changes scoring
Based on microscopic observation shown the histopathology figure of the
damage of caecum in inoculation oocyst E. tenella after serum intravenal injection
and control group (PO) without serum intravenal injection which were
haemoraghee, inflammation and vili rupture. Histopathology scoring which shown
in every treatment after tabulated then analysed using Kruskall-Wallis. The
Kruskall-Wallis data is shown in the table below :
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Table 4.3 The average hemorrhage scoring in control group and treatment groups (P1,P2,P3)
Treatments Hemorrhage ( Means ± SD) P0 2.04 b ± 0.49800 P1 1.24 a ± 0.21909 P2 1.72 b ± 0.30332 P3 2.08 b ± 0.46043
Based on data analysis using Kruskall-Wallis shows that there is a
significant differences (p<0.05) for hemorrhage. Results shows that P1 was
significantly different (p<0.05) from other treatments. Meanwhile there was no
significant difference (p>0.05) between P0 and P2 either P3 in hemorrhage
between control group and treatment group (P1,P2,P3) (p<0,05). It can be seen in
Figure 4.2, the lowest percentage of hemorrhage lesion was P1. It was followed
by P2 and P3 respectively, while the highest percentage was P0.
Table 4.4 The average inflammation scoring in control group and treatment groups (P1,P2,P3)
Treatments Inflamation ( Means ± SD) P0 3.72 b ± 0.22804 P1 2.56 a ± 0.80498 P2 2.92 a ± 0.33446 P3 3,00 a ± 0.63536
Based on data analysis using Kruskall-Wallis shows that there is a
significant difference (p<0.05) for inflammation. Results shows that all treatments
(P1,P2,P3) was significantly different (p<0.05) between control group (P0). It can
be seen in Figure 4.2, the highest percentage was P0.
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Table 4.6 The average vili rupture scoring in control group and treatment groups (P1,P2,P3)
Treatments Vili rupture (Means ± SD) P0 3.06 b± 0.66933 P1 1.08 a± 0.41473 P2 2.06 ab ± 1.40461 P3 1.36 a± 0.57271
Based on data analysis using Kruskall-Wallis shows that there is a
significant difference (p<0.05) for vili rupture. Results shows that P1 and P2 was
significantly different (p<0.05) between P0. Meanwhile there was no significant
difference (p>0.05) between P3 and P0. Kruskall-wallis test using SPSS for
histopathological changes can be seen on appendix 1.
s 4 c 3 o Inflammation r 2 i Haemorrage 1 n villi rupture g 0 P0 P1 P2 P3
Figure 4.1. Average scoring of histopathology changements in inflamation , haemorrage and vili rupture between control group (P0) and treatment group (P1,P2,P3).
4.3.2 Figure of histopathological changes chicken caecum
Figure of histopathology chicken between group control (P0) which is not
administrated by serum and challenged with 80000 sporozoites doses and group
treatment which is administrated by serum and challenged with various doses of
sporozoites. The figure of histopathology shown the severity per each treatment in
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inflammation ,hemorrhage and villi rupture caused by inoculation of sporozoites
E. tenella.
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, magnification 100x E.tenella inoculation sporozoites sporozoites inoculation
P0 Caused in Inflammation 4.2. Figure
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, magnification 100x E.tenella inoculation sporozoites sporozoites inoculation Inflammation in P1 Caused P1 Caused in Inflammation Figure 4.3 . Figure
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, magnification 100x E.tenella inoculation sporozoites sporozoites inoculation Inflammation in P2 Caused P2 Caused in Inflammation Figure 4.4 . Figure
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, magnification 100x E.tenella inoculation sporozoites sporozoites inoculation Inflammation in P3 Caused P3 Caused in Inflammation
4.5 . Figure
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, magnification 100x E.tenella inoculation sporozoites sporozoites inoculation Haemoraghe in P0 Caused Caused P0 in Haemoraghe
4.6 . Figure
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, magnification 100x
E.tenella
sporozoites inoculation
gure 4.7. Haemoraghe in P1 Caused Caused P1 in Haemoraghe 4.7. gure
Fi
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, magnification 100x
E.tenella
sporozoites inoculation
Figure 4.8. Haemoraghe in P2 Caused Caused P2 in Haemoraghe 4.8. Figure
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, magnification 100x
E.tenella
sporozoites inoculation
Figure 4.9. Haemoraghe in P3 Caused Caused P3 in Haemoraghe 4.9. Figure
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, magnification 100x E.tenella
sporozoites inoculation P0 Caused in Rupture Vili 4.10. Figure
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, magnification 100x
E.tenella
inoculation sporozoites sporozoites inoculation
P1 Caused in Rupture Vili 4.11. Figure
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, magnification 100x
E.tenella
sporozoites inoculation P2 Caused in Rupture Vili 4.12. Figure
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, 100x. magnification
E.tenella
Figure 4.13. Vili rupture in ,P3 caused by inoculation in ,P3 sporozoites 4.13. Vilicaused Figure rupture
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CHAPTER 5 DISCUSSION
5.1 Clinical Symptomps
The most pathogenic species, E. tenella, provokes a haemorrhagic diarrhea in
young chickens, leading to a loss of weight and appetite, resorption problems,
bacterial secondary infections and often also to the bird’s death (Chauhan and
Roy, 2007; Mehlhorn, 2008; Shirley et al., 2007).
Chicken from the control group shown significant clinical symptomps. On the
first-3rd day post inoculation, the chickens haven’t show any symptoms. The
chickens look healthy with a high appetite. On the 4th day post inoculation, the
chickens started to show symptomps such as a little fatigue, blody and liquid
diarrhea. On the 5-6th day, chickens shown a more sever clinical symptoms. The
chickens thirst increased and the amount of blood that came out with the faeces
were a lot. In every treatment groups (P1,P2,P3) didn’t show any clinical
symptoms. Chickens were healthy and exuberant, high appetite and normal thirs,
and normal faeces consistency.
Chickens in P1 seem to be exuberant with high appetite and normal thirst
rate, no feather loss, no bloody diarrhea and normal faeces consistency. In P2,
chickens also seem normal, normal appetite and thirst rate, a few chickens have a
little bit liquidy faeces but the rest has normal consistency. In P3, chickens seem
to be exuberant, low appetite and thirst rate compared to P1 and P2, sometimes
left over of feed and drink can be found,some has liquidy faeces consistency but
no bloody diarrhea is found compared to P0. P1,P2 and P3 group give protective
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immunity to the chickens. The existence of protective immunity in chickens body
made the chickens resistant to E. tenella (Iskandar et al., 2006).
5.2 Oocyst Production
5.2.1 Daily production of oocyst
The result of data analysis in this research which has been done in daily
production of oocyst shows there was a significant difference between group
control (P0) which is not given serum before inoculated with sporozoites of E.
tenella and group treatment (P1,P2,P3) which were given serum before incolated
with sporozoites of E. tenella.
5.3 Figure of histopathological changes chicken caecum
Data from caecum histopathological changes observation based on
histopathology disorder in preparat is scored based on sum of A and B, in which a
stood for distribution of development stadium of E. tenella along the caecum
while B stood damage severity level caused by E. tenella (Goodwin et al., 2012)
and according to (Suprihati et al., 2000) cited by (Ni’,matul, 2006),
histopathological disorder in preparations was assessed with 5 scores seen in the
visual field scored based on inflammation, hemorrhage and parasite stage.
Histopathology scoring which shown in every treatment after tabulated
then analysed using Kruskall-Wallis. Result from microscopic observation of
histopat preparat shown significant difference between control group and each
treatment group. Mean of hemorrhage scoring and inflammation in figure shown
that there was parasite development in P0 in several epithelial cells also the most
hemorrhage, inflammation and vili damage compared to the treatment groups.
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Result of the microscopic inflammation shown hemorrhage, inflammation, and
rupture in vili along with pararsite development in several epithelial cells in P1.
Scoring data shown that there was histopat disorder by degree of damage on
serum mucous deemed to be the lightes compared to P0, P2, and P3. Compat
caecum epithel, inflammation level and hemorrhage was still light and rupture is
seldom found in treatment group 2 shown almost clear caecum mucous damage
level in several parts such as hemorrhage and inflammation also parasite
development shown in several parts of the caecum but no damage was found on
the vili. P3 was similar to P2 but vili damage was a little clearer in several parts of
the caecum and parasite development in several parts. The condition corborated
with Styawati and Yuwono (1999) that clinical symptomps were shown when the
second generation schizon became bigger and the merozoites came out of the
epithelial cells causing damage in the mentioned epithelial cells and also the
rupture of blood vessels causing bleeding in caecum.
This things indicating that passive immunity from given serum in group
treatment 1,2,3 has been grown up well done, on the other hand there is no
immunity in group control (P0) (Rose, 1992).
Based on the 5 parameters, P1 is the most group that has a notation. So it
can be said that P1 is the treatment or sporozoit optimum infection dose that
shown the least clinical symptomps, oocyst production, hemorrhage lesion,
inflammation and villi rupture are the parameters that indicate that the chickens
infected with E.tenella with the administration of active vaccine can increase the
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chicken immunity system. With the administration of sporozoite antigen can
stimulate serum antibody that can increase the body immunity.
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CHAPTER 6 CONCLUSION
6.1 Conclusion
The result of this research could be conducted as follows:
1. Serum administration provided the formation of passive immunity in broiler
chicken
2. 2x104 sporozoite challenge is the optimal dose that can stimulate the best
immunity from admisitration of passive immunity
6.2 Suggestion
Based on the result of this study, the continue research more through is
needed to explore the potential of sporozoites for inducing protective immunity in
an effort to develop it as an effective seed vaccine candidate.
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SUMMARY
Inanda Ayu Shabrina. Passive Immunity Level in Broiler Chicken
Infected With Various Dose of Sporozoites on Coccidiosis infection. Under the
supervision of Prof. Mas’ud Hariadi, M.Phil., Ph.D,drh. as the first supervisor and
Muhammad Yunus, drh, M.Kes., Ph.D as co-supervisor.
As we know that broiler chicken is a kind of livestock that many
developed as a source of animal protein supply and the most rapid growth chicken
and also has a high influence in economical development in Indonesia.
Nowadays there are a lot of diseases that can attack chicken easily, one of
them is coccidiosis which is one of the most pathogenic disease that attack
intestinal that can causes bloody diarrhea, weight loss and cause of death.
Coocidiosis caused by Eimeria tenella. Since every flocks is at risk from
coccidiosis, all the 30 billion chickens reared worldwide annualy must be
protected by prophylactic chemotherapy with specific drugs or by vaccination.
Various efforts have been done to deal with this disease but nothing have
fully worked out until now. Preventive action using antiparasite causes resistance
and left residual substance, both in chickens and eggs. Preventive actions by
giving vaccine or serum for chickens is now being developped. Vaccine through
passive immunity provides immediate protection. This study aims to determine
the number of sporozoites dose in e tenella which affect the passive immunity
level of broiler chickens most effectively judged by the clinical symptoms, oocyst
production, and histopat view in broiler chickens. The research design that was
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uses in this research is complete randomized design. The procedures in this
research were serum collecting and serum inactivation, serum dose calculation
using spectophotometer method, experimental animal adaptation, clinical
symptoms observation, oocyst production, and histopat view.
Serum collecting was obtained from previous research. Serum obtained from
chicken blood that has been given active immunity (live vaccine) then serum
inactivation by keeping the serum in freezer temperature before being used.
Preparing chickens as research materials, administration of passive immunity and
inoculation with various dose of sporozoites, observation of research result and data
analysis. Observation in this research includes clinical symptoms description,
histopat view, and oocyst production. The data of clinical symptoms were analyzed
descriptively. Oocyst production was analyzed using anova in spss 18 for windows
and histopat scoring was analyzed using kruskall wallis test.
40 chickens divided into treatments where each group has 10 repetitions. In
control group chickens were not given serum (passive immunity) then inoculated
with 8x104 sporozoite Eimeria tenella in P1 chickens were given 0,5 cc serum from
8x104 sporozoite e tenella then inoculated with 2x104 sporozoite Eimeria tenella, in
P2 chickens were also given serum then infected with 4x10 dose, in P3 chickens
were infected with 8x104 dose sporozoite Eimeria tenella. Passive immunity from
serum administration in P2 and P3 caused resistance that is relatively equal. In P1
the resistance level given by passive immunity was the best compared to P2 and P3
so P1 is number of sporozoit dose in e tenella which the most effectively affected
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the passive immunity level in broiler chickens infected with coccidiosis with 2x104
dose of Eimeria tenella sporozoite.
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Morrison, D. A. 2009. Evolution of the Apicomplexa: where are we now? Trends Parasitol 25, 375–382
Mufasirin, dkk. 2012. Buku Ajar Ilmu Penyakit Protozoa. Universitas Airlangga. Surabaya. 174.
Murtidjo, B. A. 1987. Pedoman Beternak Ayam Broiler. Penerbit Kanisius. Yogyakarta
Nalbantoglu S, B Sari , H Cicek and Z karaer, 2008. Prevalence of Coccidian Species in The Water Buffalo (Bubalus bubalis) in The Province of Afyon. Turkey Acta Vet Brno, 77: 111-116.
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ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Neutra, M.R., E. Pringault and J.P. Kraehenbuhl, 1996. Antigen sampling across epithelial barriers and induction of mucosal immune responses. Ann, Rev. Immunol., 14: 275-00
Ni’matul, S.M. 2006. Pengaruh Infeksi Ookista Eimeriatenella Hasil Penyinaran Ultra Violet Terhadap Gambaran Patologis Sekum dan Jumlah Produksi Ookista pada Ayam Pedaging [Skripsi]. Fakultas kedokteran Hewan. Universitas Airlangga
Rahmawati, Anita. 2013. Gambaran Patologi pada Sekum Ayam Akibat Pemberian Ookista Eimeria tenella yang Telah Diatenuasi Melalui Pasase Berseri [Skripsi]. Fakultas kedokteran Hewan. Universitas Airlangga.
Anais, R., S.Gras, F.Lecaille, A.Niepceron, M.Katrib, N.C.Smith, G.Lalmanach, F.Brossier. 2012. Eimeripain a Cathepsin B-Like Cysteine Protease, Expressed throughout Sporulation of the Apicomplexan Parasite Eimeria tenella. Plos one. DOI: 10.1371/journal.pone.0031914
Rohayati ES, Julianti D, Nurcahyo W. 2011. Studi Pitaing Beberapa Disinfektan dalam Penanggulangan Koksidiosis pada Ayam. YogyakartaD. PPF KH UGM Rose, M.E, and P.L. Long, 1962. Immunity to four species of Eimeria in fowls. Immunol., 5: 79-92.
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Setyawati, S.J.A. dan Endro Yuwono, 1999. Upaya mendapatkan Kekebalan Terhadap Penyakit Koksidiosis Pada Ayam Buras Melalui Cara Vaksinasi. Laporan Penelitian Fak. Peternakan Unsoed.
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THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
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THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Apendix 1 : Kruskall wallis test using SPSS for histoplathological changes
SUMMARIZE
NPar Tests Descriptive Statistics
N Mean Std. Deviation Minimum Maximum
HEMORRHAGE 20 1.7700 .49535 1.00 2.60 INFLAMMATION 20 3.0500 .63536 1.60 4.00 VILI RUPTURE 20 1.8900 1.10924 .60 3.60 TREATMENT 20 2.5000 1.14708 1.00 4.00
Kruskal-Wallis Test
Ranks
TREATMENT N Mean Rank
P0 5 13.60
P1 5 3.90
HEMORRHAGE P2 5 10.40
P3 5 14.10
Total 20
P0 5 17.40
P1 5 7.10
INFLAMMATION P2 5 8.30
P3 5 9.20
Total 20
P0 5 16.30
P1 5 6.20
VILI RUPTURE P2 5 11.10
P3 5 8.40
Total 20 Test Statisticsa,b
HEMORRHAGE INFLAMMATION VILI RUPTURE
Chi-Square 9.659 9.617 8.209 df 3 3 3 Asymp. Sig. .022 .022 .042
a. Kruskal Wallis Test b. Grouping Variable: TREATMENT
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Summarize Case Processing Summarya
Cases
Included Excluded Total
N Percent N Percent N Percent
HEMORRHAGE * 20 100.0% 0 0.0% 20 100.0% TREATMENT INFLAMMATION * 20 100.0% 0 0.0% 20 100.0% TREATMENT VILI RUPTURE * 20 100.0% 0 0.0% 20 100.0% TREATMENT
a. Limited to first 100 cases.
Case Summariesa
HEMORRHAGE INFLAMMATION VILI RUPTURE
1 1.40 3.80 3.50
2 1.60 3.60 3.60
3 2.40 4.00 2.80
4 2.40 3.80 2.00
P0 5 2.40 3.40 3.40
N 5 5 5
Mean 2.0400 3.7200 3.0600 Total Std. .49800 .22804 .66933 Deviation
1 1.00 1.80 1.00 TREATMENT 2 1.60 3.00 .80
3 1.20 1.60 .60
4 1.20 3.00 1.60
P1 5 1.20 3.40 1.40
N 5 5 5
Mean 1.2400 2.5600 1.0800 Total Std. .21909 .80498 .41473 Deviation
1 1.60 2.60 .60 P2 2 1.80 3.00 1.60
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
3 2.20 3.00 1.00
4 1.60 2.60 3.60
5 1.40 3.40 3.50
N 5 5 5
Mean 1.7200 2.9200 2.0600 Total Std. .30332 .33466 1.40641 Deviation
1 2.00 3.40 1.00
2 1.40 2.60 .60
3 2.60 3.60 1.40
4 2.00 2.80 1.80
P3 5 2.40 2.60 2.00
N 5 5 5
Mean 2.0800 3.0000 1.3600 Total Std. .46043 .46904 .57271 Deviation
N 20 20 20
Total Mean 1.7700 3.0500 1.8900
Std. Deviation .49535 .63536 1.10924
a. Limited to first 100 cases.
NPar Tests Mann-Whitney Test Ranks
TREATMENT N Mean Rank Sum of Ranks
P0 5 7.70 38.50
HEMORRHAGE P1 5 3.30 16.50
Total 10
Test Statisticsa
HEMORRHAGE
Mann-Whitney U 1.500 Wilcoxon W 16.500 Z -2.363 Asymp. Sig. (2-tailed) .018 Exact Sig. [2*(1-tailed Sig.)] .016b
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
a. Grouping Variable: TREATMENT b. Not corrected for ties.
NPar Tests
Mann-Whitney Test Ranks
TREATMENT N Mean Rank Sum of Ranks
P0 5 6.50 32 .50
HEMORRHAGE P2 5 4.50 22.50
Total 10
Test Statisticsa
HEMORRHAGE
Mann-Whitney U 7.500 Wilcoxon W 22.500 Z -1.074 Asymp. Sig. (2-tailed) .283 Exact Sig. [2*(1-tailed Sig.)] .310b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
NPar Tests
Mann-Whitney Test
Ranks
TREATMENT N Mean Rank Sum of Ranks
P0 5 5.40 27.00
HEMORRHAGE P3 5 5.60 28.00
Total 10
Test Statisticsa
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
HEMORRHAGE
Mann-Whitney U 12.000 Wilcoxon W 27.000 Z -.108 Asymp. Sig. (2-tailed) .914 Exact Sig. [2*(1-tailed Sig.)] 1.000b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
NPar Tests
Mann-Whitney Test Ranks
TREATMENT N Mean Rank Sum of Ranks
P1 5 3.40 17.00
HEMORRHAGE P2 5 7.60 38.00
Total 10
Test Statisticsa
HEMORRHAGE
Mann-Whitney U 2.000 Wilcoxon W 17.000 Z -2.249 Asymp. Sig. (2-tailed) .025 Exact Sig. [2*(1-tailed Sig.)] .032b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
NPar Tests Mann-Whitney Test Ranks
TREATMENT N Mean Rank Sum of Ranks
P1 5 3.20 16.00
HEMORRHAGE P3 5 7.80 39.00
Total 10
Test Statisticsa
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
HEMORRHAGE
Mann-Whitney U 1.000 Wilcoxon W 16.000 Z -2.440 Asymp. Sig. (2-tailed) .015 Exact Sig. [2*(1-tailed Sig.)] .016b
a. Grouping Variable: TREATMENT b. Not corrected for ties. NPar Tests Mann-Whitney Test Ranks
TREATMENT N Mean Rank Sum of Ranks
P2 5 4.30 21.50
HEMORRHAGE P3 5 6.70 33.50
Total 10 Test Statisticsa
HEMORRHAGE
Mann-Whitney U 6.500 Wilcoxon W 21.500 Z -1.265 Asymp. Sig. (2-tailed) .206 Exact Sig. [2*(1-tailed Sig.)] .222b
a. Grouping Variable: TREATMENT b. Not corrected for ties. NPar Tests Mann-Whitney Test
Ranks
TREATMENT N Mean Rank Sum of Ranks
P0 5 7.90 39.50
INFLAMMATION P1 5 3.10 15.50
Total 10 Test Statisticsa
INFLAMMATION
Mann-Whitney U .500 Wilcoxon W 15.500 Z -2.530
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Asymp. Sig. (2-tailed) .011 Exact Sig. [2*(1-tailed Sig.)] .008b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
NPar Tests Mann-Whitney Test Ranks
TREATMENT N Mean Rank Sum of Ranks
P0 5 7.90 39.50
INFLAMMATION P2 5 3.10 15.50
Total 10
Test Statisticsa
INFLAMMATION
Mann-Whitney U .500 Wilcoxon W 15.500 Z -2.538 Asymp. Sig. (2-tailed) .011 Exact Sig. [2*(1-tailed Sig.)] .008b
a. Grouping Variable: TREATMENT b. Not corrected for ties. NPar Tests Mann-Whitney Test
Ranks
TREATMENT N Mean Rank Sum of Ranks
P0 5 7.60 38.00
INFLAMMATION P3 5 3.40 17.00
Total 10 Test Statisticsa
INFLAMMATION
Mann-Whitney U 2.000 Wilcoxon W 17.000 Z -2.220 Asymp. Sig. (2-tailed) .026 Exact Sig. [2*(1-tailed Sig.)] .032b
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
a. Grouping Variable: TREATMENT b. Not corrected for ties.
NPar Tests
Mann-Whitney Test
Ranks
TREATMENT N Mean Rank Sum of Ranks
P1 5 5.10 25.50
INFLAMMATION P2 5 5.90 29.50
Total 10
Test Statisticsa
INFLAMMATION
Mann-Whitney U 10.500 Wilcoxon W 25.500 Z -.434 Asymp. Sig. (2-tailed) .664 Exact Sig. [2*(1-tailed Sig.)] .690b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
NPar Tests
Mann-Whitney Test Ranks
TREATMENT N Mean Rank Sum of Ranks
P1 5 4.90 24.50
INFLAMMATION P3 5 6.10 30.50
Total 10
Test Statisticsa
INFLAMMATION
Mann-Whitney U 9.500
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Wilcoxon W 24.500 Z -.632 Asymp. Sig. (2-tailed) .527 Exact Sig. [2*(1-tailed Sig.)] .548b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
NPar Tests
Mann-Whitney Test
Ranks
TREATMENT N Mean Rank Sum of Ranks
P2 5 5.30 26.50
INFLAMMATION P3 5 5.70 28.50
Total 10
Test Statisticsa
INFLAMMATION
Mann-Whitney U 11.500 Wilcoxon W 26.500 Z -.217 Asymp. Sig. (2-tailed) .828 Exact Sig. [2*(1-tailed Sig.)] .841b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
NPar Tests Mann-Whitney Test
Ranks
TREATMENT N Mean Rank Sum of Ranks
P0 5 8.00 40.00
VILI RUPTURE P1 5 3.00 15.00
Total 10
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Test Statisticsa
VILI RUPTURE
Mann-Whitney U .000 Wilcoxon W 15.000 Z -2.611 Asymp. Sig. (2-tailed) .009 Exact Sig. [2*(1-tailed Sig.)] .008b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
NPar Tests
Mann-Whitney Test Ranks
TREATMENT N Mean Rank Sum of Ranks
P0 5 6.40 32.00
VILI RUPTURE P2 5 4.60 23.00
Total 10
Test Statisticsa
VILI RUPTURE
Mann-Whitney U 8.000 Wilcoxon W 23.000 Z -.946 Asymp. Sig. (2-tailed) .344 Exact Sig. [2*(1-tailed Sig.)] .421b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
NPar Tests
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Mann-Whitney Test
Ranks
TREATMENT N Mean Rank Sum of Ranks
P0 5 7.90 39.50
VILI RUPTURE P3 5 3.10 15.50
Total 10
Test Statisticsa
VILI RUPTURE
Mann-Whitney U .500 Wilcoxon W 15.500 Z -2.514 Asymp. Sig. (2-tailed) .012 Exact Sig. [2*(1-tailed Sig.)] .008b
a. Grouping Variable: TREATMENT b. Not corrected for ties. NPar Tests Mann-Whitney Test Ranks
TREATMENT N Mean Rank Sum of Ranks
P1 5 4.50 22 .50
VILI RUPTURE P2 5 6.50 32.50
Total 10
Test Statisticsa
VILI RUPTURE
Mann-Whitney U 7.500 Wilcoxon W 22.500 Z -1.054 Asymp. Sig. (2-tailed) .292 Exact Sig. [2*(1-tailed Sig.)] .310b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Mann-Whitney Test Ranks
TREATMENT N Mean Rank Sum of Ranks
P1 5 4.70 23.50
VILI RUPTURE P3 5 6.30 31.50
Total 10 Test Statisticsa
VILI RUPTURE
Mann-Whitney U 8.500 Wilcoxon W 23.500 Z -.843 Asymp. Sig. (2-tailed) .399 Exact Sig. [2*(1-tailed Sig.)] .421b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
Mann-Whitney Test Ranks
TREATMENT N Mean Rank Sum of Ranks
P2 5 6.00 30.00
VILI RUPTURE P3 5 5.00 25.00
Total 10 Test Statisticsa
VILI RUPTURE
Mann-Whitney U 10.000 Wilcoxon W 25.000 Z -.525 Asymp. Sig. (2-tailed) .599 Exact Sig. [2*(1-tailed Sig.)] .690b
a. Grouping Variable: TREATMENT b. Not corrected for ties.
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
APENDIX 2 : Kruskall-Wallis test using SPSS for daily production of oocyst
NPAR TESTS /K-W=day1 BY perlakuan(1 4) /MISSING ANALYSIS.
NPar Tests
Notes
Output Created 11 -AUG-2015 16:04:04 Comments D: Data anda.sav Active Dataset DataSet0 Filter
a. Based on availability of workspace memory.
[DataSet0] D:\nanda.sav
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Kruskal-Wallis Test
Ranks
perlakuan N Mean Rank p0 5 18.00 p1 5 8.00 day1 p2 5 8.00 p3 5 8.00 Total 20
Test Statisticsa,b
day1
Chi-Square 18.603 df 3 Asymp. Sig. .000
a. Kruskal Wallis Test b. Grouping Variable: perlakuan
NPAR TESTS /K-W=day2 BY perlakuan(1 4) /MISSING ANALYSIS.
NPar Tests
Notes
Output Created 11 -AUG-2015 16:10:20 Comments
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
D: Data anda.sav Active Dataset DataSet0 Filter
Resources Elapsed Time 00:00:00.01 Number of Cases Alloweda 112347
a. Based on availability of workspace memory.
[DataSet0] D:\nanda.sav
Kruskal-Wallis Test
Ranks
perlakuan N Mean Rank p0 5 18.00 p1 5 8.40 day2 p2 5 8.60 p3 5 7.00
Total 20
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Test Statisticsa,b
day2 Chi-Square 15.051 df 3 Asymp. Sig. .002
a. Kruskal Wallis Test b. Grouping Variable: perlakuan
NPAR TESTS /K-W=day3 BY perlakuan(1 4) /MISSING ANALYSIS.
NPar Tests
Notes
Output Created 11 -AUG-2015 16:10:53 Comments D: Data anda.sav Active Dataset DataSet0 Filter
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Processor Time 00:00:00.00 Resources Elapsed Time 00:00:00.01 Number of Cases Alloweda 112347
a. Based on availability of workspace memory.
[DataSet0] D:\nanda.sav
Kruskal-Wallis Test
Ranks
perlakuan N Mean Rank
p0 5 18.00 p1 5 7.00
day3 p2 5 7.00 p3 5 10.00
Total 20
Test Statisticsa,b
day3
Chi-Square 15.932 df 3 Asymp. Sig. .001
a. Kruskal Wallis Test b. Grouping Variable: perlakuan
NPAR TESTS /K-W=day4 BY perlakuan(1 4) /MISSING ANALYSIS.
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
NPar Tests
Notes
Output Created 11 -AUG-2015 16:11:54 Comments D: Data anda. sav Active Dataset DataSet0 Filter
Resources Elapsed Time 00:00:00.01 Number of Cases Alloweda 112347
a. Based on availability of workspace memory.
[DataSet0] D:\nanda.sav
Kruskal-Wallis Test
Ranks
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
perlakuan N Mean Rank p0 5 18.00 p1 5 7.00 day4 p2 5 10.00 p3 5 7.00 Total 20
Test Statisticsa,b
day4 Chi-Square 15.948 df 3 Asymp. Sig. .001
a. Kruskal Wallis Test b. Grouping Variable: perlakuan
NPAR TESTS /K-W=day5 BY perlakuan(1 4) /MISSING ANALYSIS.
NPar Tests
Notes
Output Created 11 -AUG-2015 16:12:13 Comments D: Data anda.sav Active Dataset DataSet0 Filter
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
User-defined missing values are Definition of Missing treated as missing. Missing Value Handling Statistics for each test are based on all Cases Used cases with valid data for the variable(s) used in that test. NPAR TESTS Syntax /K-W=day5 BY perlakuan(1 4) /MISSING ANALYSIS. Processor Time 00:00:00.02 Resources Elapsed Time 00:00:00.01 Number of Cases Alloweda 112347
a. Based on availability of workspace memory.
[DataSet0] D:\nanda.sav
Kruskal-Wallis Test
Ranks
perlakuan N Mean Rank
p0 5 18.00
p1 5 6.50
day5 p2 5 8.20 p3 5 9.30 Total 20
Test Statisticsa,b
day5 Chi-Square 14.388 df 3 Asymp. Sig. .002
a. Kruskal Wallis Test
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
b. Grouping Variable: perlakuan
NPAR TESTS /K-W=day6 BY perlakuan(1 4) /MISSING ANALYSIS.
NPar Tests
Notes
Output Created 11 -AUG-2015 16:12:37 Comments D: Data anda.sav Active Dataset DataSet0 Filter
a. Based on availability of workspace memory.
[DataSet0] D:\nanda.sav
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Kruskal-Wallis Test
Ranks
perlakuan N Mean Rank p0 5 18.00 p1 5 7.80 day6 p2 5 8.00 p3 5 8.20 Total 20
Test Statisticsa,b
day6
Chi-Square 13.664 df 3 Asymp. Sig. .003
a. Kruskal Wallis Test b. Grouping Variable: perlakuan
NPAR TESTS /K-W=day7 BY perlakuan(1 4) /MISSING ANALYSIS.
NPar Tests
Notes
Output Created 11 -AUG-2015 16:13:06 Comments
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
D: Data anda.sav Active Dataset DataSet0 Filter
Resources Elapsed Time 00:00:00.01 Number of Cases Alloweda 112347
a. Based on availability of workspace memory.
[DataSet0] D:\nanda.sav
Kruskal-Wallis Test
Ranks
perlakuan N Mean Rank p0 5 18.00 p1 5 7.80 day7 p2 5 7.60 p3 5 8.60
Total 20
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Test Statisticsa,b
day7 Chi-Square 12.934 df 3 Asymp. Sig. .005
a. Kruskal Wallis Test b. Grouping Variable: perlakuan
MEANS TABLES=day1 day2 day3 day4 day5 day6 day7 BY perlakuan /CELLS MEAN COUNT STDDEV.
Means
Notes
Output Created 11 -AUG-2015 16:16:47 Comments D: Data anda.sav Active Dataset DataSet0 Filter
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
MEANS TABLES=day1 day2 day3 Syntax day4 day5 day6 day7 BY perlakuan /CELLS MEAN COUNT STDDEV. Processor Time 00:00:00.03 Resources Elapsed Time 00:00:00.05
[DataSet0] D:\nanda.sav
Case Processing Summary
Cases Included Excluded Total
N Percent N Percent N Percent
day1 * perlakuan 20 100.0% 0 0.0% 20 100.0% day2 * perlakuan 20 100.0% 0 0.0% 20 100.0% day3 * perlakuan 20 100.0% 0 0.0% 20 100.0% day4 * perlakuan 20 100.0% 0 0.0% 20 100.0% day5 * perlakuan 20 100.0% 0 0.0% 20 100.0% day6 * perlakuan 20 100.0% 0 0.0% 20 100.0% day7 * perlakuan 20 100.0% 0 0.0% 20 100.0%
Report
perlakuan day1 day2 day3 day4
Mean 32140.0000 156600.0000 169400.0000 241600.0000
p0 N 5 5 5 5 Std. Deviation 294 0.74820 96756.39514 44461.21906 88976.40137 Mean .0000 2200.0000 .0000 .0000 p1 N 5 5 5 5 Std. Deviation .00000 4919.34955 .00000 .00000 Mean .0000 3600.0000 .0000 7000.0000 p2 N 5 5 5 5 Std. Deviation .00000 8049.84472 .00000 9746.79434 Mean .0000 .0000 9800.0000 .0000 p3 N 5 5 5 5 Std. Deviation .00000 .00000 13423.85936 .00000
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S ADLN-PERPUSTAKAAN UNIVERSITAS AIRLANGGA
ADLN – PERPUSTAKAAN UNIVERSITAS AIRLANGGA
Mean 8035.0000 40600.0000 44800.0000 62150.0000 Total N 20 20 20 20 Std. Deviation 14342.18199 81931.55038 76931.00121 113992.73984
Report
perlakuan day5 day 6 day7 Mean 370400.0000 314400.0000 352000.0000
p0 N 5 5 5 Std. Deviation 126264.80111 138408.09225 155104.80328 Mean .0000 1080.0000 4200.0000 p1 N 5 5 5 Std. Deviation .00000 2414.95342 9391.48551 Mean 4000.0000 2600.0000 4000.0000 p2 N 5 5 5 Std. Deviation 8944.27191 5813.77674 8944.27191 Mean 5600.0000 4400.0000 4200.0000 p3 N 5 5 5 Std. Deviation 7956.12971 9838.69910 6572.67069 Mean 95000.0000 80620.0000 91100.0000
Total N 20 20 20
Std. Deviation 173214.50043 152445.75774 1702 73.43335
THESIS PASSIVE IMMUNITY LEVEL ... INANDA AYU S..
THESIS PASSIVE IMMUNITY LEVEL... INANDA AYU S