EFFICACY TEST OF ANTIBIOTICS ON NATURALLY DISEASED CLIMBING PERCH Anabas testudineus A THESIS BY KAZI ASHEQUE MAHAMUD EXAMINATION ROLL NO. 10 Fish Aqua JD-34M SEMESTER: JULY-DECEMBER, 2011 REGISTRATION NO: 32621 SESSION: 2005-2006 MASTER OF SCIENCE (M. S.) IN AQUACULTRE DEPARTMENT OF AQUACULTURE BANGLADESH AGRICULTURAL UNIVERSITY MYMENSINGH NOVEMBER, 2011
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EFFICACY TEST OF ANTIBIOTICS ON NATURALLY DISEASED CLIMBING PERCH Anabas testudineus
A THESIS
BY
KAZI ASHEQUE MAHAMUD
EXAMINATION ROLL NO. 10 Fish Aqua JD-34M
SEMESTER: JULY-DECEMBER, 2011
REGISTRATION NO: 32621 SESSION: 2005-2006
MASTER OF SCIENCE (M. S.)
IN
AQUACULTRE
DEPARTMENT OF AQUACULTURE
BANGLADESH AGRICULTURAL UNIVERSITY
MYMENSINGH
NOVEMBER, 2011
2 2
EFFICACY TEST OF ANTIBIOTICS ON NATURALLY DISEASED CLIMBING PERCH Anabas testudineus
A THESIS
BY
KAZI ASHEQUE MAHAMUD
EXAMINATION ROLL NO. 10 Fish Aqua JD-34M SEMESTER: JULY-DECEMBER, 2011
REGISTRATION NO: 32621
SESSION: 2005-2006
Submitted to the
Department of Aquaculture
Bangladesh Agricultural University, Mymensingh
In partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE (M. S.)
IN
AQUACULTURE
NOVEMBER, 2011
3 3
EFFICACY TEST OF ANTIBIOTICS ON NATURALLY DISEASED CLIMBING PERCH Anabas testudineus
A THESIS
BY
KAZI ASHEQUE MAHAMUD
EXAMINATION ROLL NO. 10 FishAqua JD-34 M SEMESTER: JULY-DECEMBER, 2011
REGISTRATION NO: 32621
SESSION: 2005-2006
Approved as to style and content by:
……………………….................... ………………………………………. (Prof. Dr. M. Mamnur Rashid) (Prof. Dr. Kirtunia Juran Chandra) Supervisor Co- Supervisor
................................................
(Prof. Dr. Md. Ali Reza Faruk)
Chairman, Examination Committee
And Head, Department of Aquaculture
Bangladesh Agricultural University Mymensingh
NOVEMBER, 2011
4 4
ABSTRACT
A study was conducted to examine the effects of different antibiotics against bacterial
infection in climbing perch Anabas testudineus. Naturally diseased fish were collected
from different places of Mymensingh region. Before starting antibiotic trial, it was
confirmed that the collected diseased fish Anabas testudineus were infected with bacteria
by primary characterization in laboratory condition. Bacteria infected Anabas testudineus
showed haemorrhage and ulcerative lesions over the body, especially near head and caudal
region, exophthalmia and dark body coloration. A total of 27 diseased fish were used for
this experiment. Three antibiotics: Oxysentin 20% (oxytetracycline HCL BP), Acimox
(amoxicillin Tri hydrate BP), Oxy-D VET (Oxytetracycline 20% and + Doxycycline 10%)
were used in separate nine aquaria at lower, recommended and higher dose respectively.
Dose of Oxysentin 20% (oxytetracycline HCL BP) were given as 25 g, 35 g, 45 g/100 Kg
body weight. Dose of Acimox (amoxicillin Tri hydrate BP), were given as 4 g, 5 g, 7.5
g/15 Kg body weight. Dose of Oxy-D VET (Oxytetracycline 20% and + Doxycycline
10%) were given as .80 g, 1 g, 1.5 g/4 Kg body weight. Water was exchanged and
artificial feed was supplied regularly. The antibiotics trial was conducted for 10 days.
Among the three antibiotic Oxysentin 20% (oxytetracycline HCL BP) and Acimox
(amoxicillin Tri hydrate BP), treatment at higher dose showed good result where 100%
fish were recovered. But combined effect of Oxy-D VET (oxytetracycline 20% and +
doxycycline 10%) at recommended dose treatment showed the best result where 100%
fish were recovered.
5 5
ACKNOWLEDGEMENTS The author always likes to bow his head to Almighty Allah who enabled him to pursue the research work and writing up this thesis for the degree of Master of Science (M.S.) in Aquaculture. The author sincerely expresses his deep sense of gratefulness, indebtedness and profound respect to his honorable teacher and supervisor Dr. M. Mamnur Rashid, Professor, Department of Aquaculture, Bangladesh Agricultural University, Mymensingh for his scholastic supervision, valuable suggestions, sympathetic co-operation, helpful advice, constructive criticism, painstaking correction of the manuscript and affectionate feeling at all stages of this study period, research work and preparation of this thesis. The author also finds great pleasure to express his sincere appreciation and immense indebtedness to his co-supervisor, Prof. Dr. Kirtunia Juran Chandra, Department of Aquaculture, Bangladesh Agricultural University, Mymensingh for his sympathetic consideration, valuable suggestions, painstaking correction of the manuscript and constructive criticism throughout the research period and the thesis work. The author would like to acknowledge his sincere gratitude to Professor Dr. Md. Ali Reza Faruk Head, Department of Aquaculture, Bangladesh Agricultural University, Mymensingh for his kind co-operation, valuable instruction and cordial support during this research work. The author feels it an opportunity to express his gratitude, indebtedness and profound respect to his honorable teachers, Prof. Dr. Md. Mohosin Ali, Prof. Dr. Monoronjan Das, Prof. Dr. Md. Bazlur Rashid Chowdhury, Prof. Dr. S. M. Rahmatullah, Prof. Dr. Md. Ruhul Amin, Prof. Dr. Ahsan Bin Habib, Prof. Dr. Gias Uddin Ahmed, Prof. Dr. M. A. Salam, Dr. Mohammad Mahfuzul Haque and Mr. Md. Sazzad Hossain for their valuable suggestions and sympathetic co-operation throughout the research period. The author expresses his gratefulness to his senior researcher Mr. Md. Taufiqual Islam for his sympathetic co-operation and helpful advice throughout the research period. Special thanks are for Eon Pharmaceutical Ltd. for their liberal gratitude by rendering their products during the experimental trail. The author would like to express special thanks to his friends Titas, Dollar, Ranga mamun, mizan, Roni, jahid, Lazu, Robi, Rakib, and Istiak for their helpful suggestions and sympathetic co-operation throughout the research period. The author can never repay the debt to his beloved parents Mr.Kazi Habibullah and Mahamuda Begum, Raju fupa, Younger brother Kazi Thasin Mahamud, and all most Irin for their sacrifice, blessings and encouragements during his study at BAU. The Author November, 2011
6 6
CONTENTS
CHAPTER PAGE
ABSTRACT i
ACKNOWLEDGEMENTS 6
CONTENTS 6
LIST OF TABLES 6
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 5
3 MATERIALS AND METHODS 10
3.1 Experimental naturally diseased fish 10
3.2 Primary determination of disease 10
3.3 Acclimatization of fish 10
3.4 Selected group of antibiotics 11
3.5 Trial of antibiotics 12
3.6 Experimental setup 12
3.7
3.8
Number of fish for each aquarium
Calculation and application of antibiotics
14
16
4 RESULTS 22
4.1 Temperature of the recycle system 22
4.2
4.3
Gross clinical features of the diseased fish
Improved condition of the treated fish
22
27
5 DISCUSSION 31
6 SUMMARY AND CONCLUSION 35
REFERENCES 37
7 7
LIST OF TABLES SL. NO.
TITLE PAGE
1 Water temperature of recycle system during the antibiotic trial 23
2 Antibiotic effects on Anabas testudineus infected with bacteria 26
8 8
LIST OF FIGURES
SL. NO.
TITLE PAGE
1 Layout of the recirculatory system used for expremental infections of koi by Aeromonas hydrophila. A, five metallic drums; B, motor and overhead tank; C, downward pipe ventilated to each aquarium and the collection pipe; D, ultra-violet tube light complex
13
2 Fish are being treated with antibiotic (Oxysentin 20%), at lower dose, recommended dose, and higher dose.
15
3 Fish are being treated with antibiotic (Acimox), at lower dose, recommended dose, and higher dose.
15
4 Fish are being treated with (Oxy-D Vet), at lower dose, recommended dose, and higher dose.
15
5 Bacteria infected Anabas testudineus showing (↑) hemorrhage on the body surface.
24
6 Bacteria infected Anabas testudineus showing (↑) hemorrhagic lesion on the pectoral region.
24
7 Bacteria infected Anabas testudineus showing (↑) hemorrhagic on the caudal region
24
8 Bacteria infected Anabas testudineus showing (↑) ulcerative lesions and darkening body color.
24
9 Bacteria infected Anabas testudineus showing (↑) ulcerative lesion on the head.
11 Bacteria infected Anabas testudineus showing (↑) ulcerative lesion on the caudal region.
25
12 Bacteria infected Anabas testudineus showing (↑) irregular caudal fin rays. 25 13 Photographs of cured Anabas testudineus after treated with Oxysentin 20%
at recommended dose. 28
14 Photographs of cured Anabas testudineus after treated with Oxysentin 20% at higher dose.
28
15 Photograph of cured Anabas testudineus from exophthalmia after treated with Oxysentin 20% higher dose.
28
16 Photographs of cured Anabas testudineus after treated with Acimox at recommended dose.
29
17 Photographs of cured Anabas testudineus after treated with Acimox at higher dose.
29
18 Photograph of cured Anabas testudineus from haemorrhage after treated with Acimox at higher dose.
29
19 Photographs of cured Anabas testudineus after treated with Oxy-D Vet at recommended dose.
30
20
21
Photographs of cured Anabas testudineus after treated with Oxy-D Vet at higher dose. Photographs of cured Anabas testudineus from ulcerative lesions after treated with Oxy-D Vet at recommended dose.
30
30
9 9
CHAPTER 1
INTRODUCTION
10 10
CHAPTER 1
INTRODUCTION
Aquaculture in Bangladesh is under heavy expansion. In aquaculture as in all food
production sectors, one of the external imputes required for successful fish
production is aqua medicine. Aqua-medicines are indeed essential ingredients to
successful aquaculture. Aqua medicines are also important component in health
management of aquatic animal, pond construction, soil and water management,
improving natural aquatic productivity, transportation of live fish, feed formulation,
manipulation of reproduction, growth promotion and processing value
enhancement of final product (Alderman et al. 1994, GESAMP, 1997) etc. With the
expansion of aquaculture in Bangladesh, there has been an increasing trend in using
more medicine in aquatic animal health management. Aqua-medicine are indeed
essential ingredients to successful aquaculture, which has been used in various
forms for centuries (Subainghe et al. 1996).
Antibiotics are very useful additions to any fish-health manager's toolbox, but they
are only tools and not 'magic bullets’. Antibiotics, by themselves, do not cure a fish.
Antibiotics merely control the population growth of bacteria in a fish long enough
for its immune system to eliminate them.
The vast majority of oxytetracycline and oxolinic acid provided is likely to leave
the farm as particulate wasts because of feed wastage and poor digestive absorption
of these drugs (Cravedi et al. 1987).
The technical term for the branch of medical science that determines all of these
variables is 'pharmacokinetics'; defined as the study of how drugs are absorbed by,
distributed within, chemically altered within, and eventually excreted by the body
(in this case, the body of a fish). The pharmacokinetics of the antibiotic,
oxytetracycline (OTC), were conducted to improve oral dosing efficacy and safety
for OTC use in treating shrimp Litopenaeus setiferus (Lou Ann Reed et al. 2006).
11 11
Antibiotics have been applied in aquaculture for over 50 years for treating bacterial
infection in fish. The early use of antibiotics was the use of sulphonamides in the
treatment of furanculosis in trout and the tetracycline against a range of gram
negative pathogen (Ali, 2008). Antibacterial chemotherapy is applied in
aquaculture throughout the world. In many countries there is considerable
prophylactic use of antibacterials.
Thus, many problems have been associated with use of aqua medicines. Many
marginal farmers face the lack of efficiency of aqua-medicine. They are not truly
benefited through the using the recommended dose of aqua-medicine from different
pharmaceutical companies. On the other hand, if the dose of antibiotic is too low or
treatment time is too short, the bacteria will not be killed or weakened enough for
the immune system of the fish to remove them, and this greatly increases the risk of
the bacteria developing resistance to the antibiotic. When bacteria become resistant
to a specific antibiotic, even high concentrations of that drug will not be effective.
Decreased efficacy has been documented in many antimicrobial drugs regardless of
their mechanism of action (Beverly A. Dixon, 1994).
Forty pharmaceutical companies have been recorded to marketed their products.
Most of the products have been imported from different countries like USA,
Thailand, Malaysia, Belgium and China (Islam, 2010). The effectiveness of the
aqua-medicines were might being reduced due to mixing of other fine ingredients
by the local traders.
Poor farming practices, including those that cause water pollution and other
negative impacts on the environment as well as the over use of chemicals and
antibiotics are bad news. So different concentrations of the selected antibiotic need
to be tested against various bacteria in order to determine exact concentrations
required against these bacteria.
12 12
The purpose of these measurements is to determine duration of the antibiotic stays
active in the body of the fish and whether or not the concentration in the body is
high enough to kill or inhibit bacteria.
Since the launch of aqua medicine few researches have yet been undertaken. In this
point of view, it is necessary to evaluate the risks associated with aqua-medicine
and establish the standard dose and dosage of aqua-medicine which needs
examination.
To fulfill the above desires, the objectives of the present study were therefore:
To determine the actual efficacy of some selected antibiotics;
To know the exact dose, dosage and method of their application; and
finally
To identify the problems associated with their recommended doses.
13 13
CHAPTER 2 REVIEW OF LITERATURE
14 14
CHAPTER 2
REVIEW OF LITERATURE A number of diversified researches regarding the use of chemotherapeutics used in
aquatic animal's health management have been carried out world-wide. The
following information, relevant to the present study was briefly reviewed:
Adkinson (1980) reported that although allergic reaction caused by antibiotic
residues in food are that of great public concern. They found that, in general, the
incidence of allergic reaction following ingestion of antibiotic resides in food
animal were very low.
Lipton (1991) studied the effect of antibiotic compounds on the growth inhibition
of fish pathogen Aeromonas hydrophila isolated from the haemorrhagic lesions of
Labeo rohita. He found that among the ten antibiotics, gentamycin, tetracycline,
streptomycin, penicillin and neomycin inhibited the growth of the bacteria.
Antibiotics gentamycin, streptomycin and tetracycline were effective at 10 g/ml.
Tetracycline was effective at 20 g/ml and gentamycin, neomycin and streptomycin
at 50 g/ml for A. hydrophila.
Hansen et al. (1992) observed that oxytetracycline, oxolinic acid, and flumequine
were the most frequently used antibacterial agents in the treatment of marine
farmed fish in Norway. These substances were supplied with the food pellets and a
substantial amount ended up in the sediment under the net pens. The effects of
these antibacterial agents on the microbial community in the sediment were
experimentally examined in tanks containing sediment. During the first 20 days of
the experiment, approximately one-third of the antibacterial agents disappeared
from the sediments. The number of bacteria in all treated sediments decreased to
50–67% of the numbers in the control sediment 2 days after medication.
15 15
Ahmed and Tan (1992) reported that the use of 16.67 mg/l of tetracycline was
effective for the wound healing of the epidermis of Clarias macrocephalus within
28 days.
Dixon (1994) suggested that antibiotic resistance by bacterial fish pathogens was
reported in all areas of aquaculture from warm water to coldwater, and freshwater
to marine environments. Decreased efficacy was documented in many
antimicrobial drugs regardless of their mechanism of action. Alternatives to the
currently used antimicrobial therapies were being evaluated for use in aquaculture,
particularly the new fluoroquinolones and the third generation cephalosporins.
Smith et al. (1994) observed that oxytetracycline was one of the most widely used
antibacterials in aquaculture worldwide. The vast majority of oxytetracycline
supplied in mediated feed can be found in hatchery effluent at concentrations that
account for nearly all of the drugs supplied.
DePaola et al. (1995) investigated the effect of oxytetracycline-medicated feeds on
antibiotic resistance in gram-negative bacteria from fish intestines and water in
catfish ponds. Percentages of tetracycline-resistant bacteria in catfish intestines
obtained from medicated ponds increased significantly after 10 days of treatment.
oxytetracycline treatment did not affect the distribution of bacterial species in the
fall but may have accelerated a shift toward greater prevalence of members of the
family Enterobacteriaceae in the spring. Multiple antibiotic resistances did not
appear to be elicited by oxytetracycline treatment.
Prasad et al. (1996) investigated the effect of five different antibiotics on EUS
affected fish and found that chloramphenicol and oxytetracycline would be
effective drug in curing the EUS lesion, tetracycline and streptomycin were found
to be less effective in curing the ulcers.
16 16
Inglis (1996) reported that anti-bacterial chemotherapy has been applied in
aquaculture for over 50 years, with early attempts to use sulphonamides in the
treatment of furunculosis in trout and the tetracycline against a range of gram
negative pathogens.
Singh and Sing (1997) obtained seven isolates of Edwardsiella tarda and showed
that all the isolates were resistant to colistin and gentamicin, but sensitive to
ciprofloxacin, chlorarnphenicol, nalidixic acid, nitrifurantoin, ofloxacin and
streptomycin.
Haque et al. (1997) studied on the Minimal Inhibitory Concentration (MIC) and
Minimal Bactreicidal Concentration (MBC) of three commonly used antibiotics
and found that most of the antibiotics could not inhibit organisms under the range
of concentration tested, amphicillin ( 2 to 64 mcg/ml), amoxicillin (I to 32 mcg/ml)
and tetracycline ( I to 32 mcg/ml).
.
Tafalla (1999) suggested the use of oxytetracycline (OTC) was one of the most
frequently used antibiotics in aquaculture, although negative side-effects were
reported in some cases. Although cell viability did not decrease after in vitro
exposure, head kidney macrophage respiratory burst and phagocytosis were
inhibited by the in vitro treatment, and were dose-dependent.
Mastan and Qureshi (2001) examined the effect of different antibiotics on EUS
affected fish Channa stariatus (Bloch) and found that at 20 ppm dose healing effect
started to take place after 4,6,and 6 days in case of chlorampenical, oxytetracycline
and ciprofloxacine exposed fishes respectively. Whereas, in the control group, the
natural healing action was noticed after 2 weeks.
17 17
Miranda et al. (2002) observed the bacterial resistance to oxytetracycline in Chilean
salmon farming. Oxytetracycline was frequently used in Chile to prevent and
control bacterial pathogens in salmon farming, as well as the level of resistance of
selected strains was investigated. Resistance levels of selected strains isolated in
media containing antibiotic were determined using an agar plate dilution method.
One hundred and three resistant Gram-negative isolates represented the
oxytetracycline resistant bacterial population.
Chowdhury et al. (2003) reported that the antibiotic, renamycin (oxytetracyline)
had positive effect against bacterial, infection at a dose of 50 mg/Kg body wt/day
applying for days and 80-90% fish were recovered under laboratory condition.
Bruun et al. (2003) suggested that the medication effect of oxytetracycline on
groups of rainbow trout fry experimentally infected with three strains of
Flavobacterium psychrophilum was investigated. The infection model was based
on intraperitoneal injection of the pathogen and treatment was done using
medicated feed resulting in 100 mg oxytetracycline/ Kg fish for 10 days.
Rocca et al. (2004) carried out the single dose administration (trial 1): serum and
tissue concentrations of amoxicillin (AMX) were investigated in sea bream (Sparus
aurata L.) kept in seawater at 22° C and 32‰ of salinity. Amoxicillin was given
intravenously (i.v.) at 40 mg/ Kg b.w. or orally (p.o.) at 80 mg/ Kg b.w. Different
formulations (conventional, micronized and microencapsulated AMX) were
assayed in seabream at 24–26 °C after a 5-day period on medicated diet at the dose
of 80 mg/kg b.w./day (trial 3) to verify if the nonconventional forms could improve
the tissue distribution of AMX after in-feed administration.
Reed et al. (2006) reported that the pharmacokinetics of the antibiotic,
oxytetracycline (OTC), were examined following oral dosing in the white shrimp,
Litopenaeus setiferus. These studies were conducted to improve dosing efficacy
and safety for OTC use in treating shrimp. Both pharmacokinetics and
18 18
physicochemical properties were studied under conditions simulating a marine
environment. While single dose studies with individual shrimp suggested that
OXTC was the preferred form of OTC, multiple dose studies with multiple shrimp
showed that practically there were little differences in therapeutic efficacy or
depuration times between the two forms of OTC.
Islam (2010) examined the effects of different chemotherapeutics against
Aeromonas hydrophila infection in climbing perch Anabas testudineus. Diseased
fish were collected from different places of Mymensingh region. Infected Anabas
testudineus showed haemorrhage and ulcerative lesions over the body, especially
near the mouth, head, and caudal region. Internally, kidney, liver, and spleen were
swollen and enlarged. The chemotherapeutic trial was conducted for 15 days. Only
antibiotic treatment showed the best result where 100% fish were recovered. Single
CuSO4 treatment also showed good result where 80% fish were recovered.
DeCew (2011) tested antibiotics for their toxicity and efficacy in adult spring
chinook salmon (Oncorhynchus tshawytscha) infected with bacterial kidney disease
and furunculosis. oxytetracycline-HCl was not toxic and it effectively controlled
both diseases, producing a three-fold increase in adult survival and production of
viable eggs. Mandible and fin teratogenesis occurred in progeny of adults treated
with the above antibiotic complex, but could be reduced by providing a 32 day
interim between injection and spawning.
[
19 19
CHAPTER 3 MATERIALS AND METHODS
20 20
CHAPTER 3
MATERIALS AND METHODS
3.1 Experimental naturally diseased fish
Studies were conducted to investigate, in laboratory condition, the effect of
antibiotics against bacteria causing infectious diseases in climbing perch Anabas
testudineus, collected from various places of Mymensingh district. In total 27
naturally diseased fish were collected.
3.2 Primary determination of disease
For primary detection of any bacterial disease following general disease symptoms
were investigated:
1. Presence of any external hemorrhage.
2. Presence of any superficial and or ulcerative lesion.
3. Darkening of body colour.
4. Condition of any exophthalmia.
5. Loss or rot of any fin rays.
6. Condition of any scale loss.
7. Any cork screw or vertical swimming.
8. Any abnormal feeding tendency.
9. Any sluggish movement or frequent rest.
Secondly, the internal organs of representative fish were homogenized and 100 µl
were plated and incubated to observe bacterial growth, for confirmation of the
infection.
21 21
Trade Name: Oxysentin 20%
Composition: Oxytetracycline HCl BP
Company Name: Novartis
Recommended Dose: 28-40 g/100 Kg fish
for 10 days
Trade Name: Acimox
Composition: Amoxicillin Tri hydrate BP
Company Name: ACI
Recommended Dose: 5 g/15 Kg fish for 10
days twice daily
Trade Name: Oxy-D Vet
Composition: Oxytetracycline 20% and
Doxycycline 10%
Company Name: EON
Recommended Dose: 1 g/4 Kg fish for 10
days twice daily
3.3 Acclimatization of fish
Above 27 naturally diseased fish were acclimatized for 3 days in laboratory
condition with recirculatory water system as mentioned later in this chapter.
3.4 Selected group antibiotics
Antibiotics were selected through personal contact with the representatives of
pharmaceutical companies and also with the pharmaceutical stores of Mymensingh.
Selected antibiotics were the following.
22 22
[3.5 Trial of antibiotics
Trials of antibiotics were conducted in a water recirculatory system in the wet
laboratory, Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh.
3.6 Experimental setup
Water recirculatory system in the wet laboratory was consisted of 12 rectangular
glass aquaria of 40 l capacity, 4 drums, 1 pump and an overhead tank. Water was
picked up into the overhead tank by pump. Freshwater from underground deep
pump was added to the recirculatory system as and when needed to fill up the loss
due to evaporation (Fig. 1).
Out of the above 12 aquaria, 9 were used for the trail. The antibiotic Oxysentin
20% treatment was given in first three aquaria and the next three aquaria were
treated with antibiotic Acimox and rest three aquaria, with Oxy-D Vet. The each
three separate aquaria were designated as Section-A, Section-B, and Section-C