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EVALUATION OF THERAPEUTIC AND PROPHYLACTIC POTENTIALS OF TOLTRAZURIL AGAINST CAECAL
COCCIDIOSIS OF CHICKENS IN BANGLADESH
A Thesis
Submitted to Bangladesh Agricultural University, Mymensingh
In partial Fulfillment of the Requirements for the Degree of
Master of Science in
Pathology
By
MD. MUSTAFA ASHRAF EXAMINATION ROLL NO. 10 VETPATH JD 01M
SEMESTER: JULY-DECEMBER 2011 REGISTRATION NO. 15819
SESSION: 1988-1989
Department of Pathology Bangladesh Agricultural University
Mymensingh
November, 2011
EVALUATION OF THERAPEUTIC AND PROPHYLACTIC POTENTIALS OF TOLTRAZURIL AGAINST CAECAL
COCCIDIOSIS OF CHICKENS IN BANGLADESH
A Thesis
Submitted to Bangladesh Agricultural University, Mymensingh
In partial Fulfillment of the Requirements for the Degree of
Master of Science in
Pathology
By
MD. MUSTAFA ASHRAF
Approved as to style and contents by
Prof. Dr. Priya Mohan Das Prof. Dr. Md. Abu Hadi Noor Ali Khan
Co-Supervisor Supervisor
Prof. Dr. Priya Mohan Das Chairman, BOS & Head Department of Pathology
Faculty of Veterinary Science Bangladesh Agricultural University
Mymensingh-2202
November, 2011
Dedicated to My Beloved Parents
ACKNOWLEDGEMENTS
All panegyrics are due to the Almighty Allah, the Supreme Authority of the Universe, Who has kindly
enabled the author to conduct the research and thesis work successfully for the degree of Master of
Science in Pathology.
The author would like to express his heartily gratefulness to National Agricultural Technology Project
(NATP), Department of Livestock Services (DLS), Dhaka, Bangladesh for financial assistance to
conduct the research work successfully.
The author would like to express his heartfelt gratitude, indebtedness and profound respect to his
honorable teacher and research supervisor Dr. Md. Abu Hahi Noor Ali Khan, Professor, Department
of Pathology, BAU, Mymensingh for his generosity, scholastic guidance, invaluable advice,
suggestions, constructive criticism, untiring help and constant inspiration throughout the course of
this research work and immense help in preparing the thesis manuscript.
The author wishes to convey his profound respect and sincere gratitude to his honorable teacher and
research co-supervisor Dr. Priya Mohan Das, Professor, Department of Pathology, BAU,
Mymensingh, for his affectionate encouragement, constructive criticism, kind co-operation, necessary
correction and instruction to complete this manuscript.
The author would like to express his immense indebtedness to Professor Dr. Md. Iqbal Hossain,
Professor Dr. Md. Abdul Baki, Professor Dr. Md. Rafiqul Islam, Professor Dr. Md. Mokbul Hossain,
Professor Dr. Md. Habibur Rahman, Professor Dr. A. S. Mahfuzul Bari, Professor Dr. Emdadul
Haque Chowdhury, DR. Rokshana Parvin, DR. Jahan Ara Begum, DR. Mohammed Nooruzzaman
and DR Munmun Pervin Department of Pathology, Bangladesh Agricultural University,
Mymensingh, for giving encouragement, advice and facilitating the lab equipments and reagents to
conduct the research work.
The author also wishes to express his gratefulness and sincere appreciation to all PhD and MS
students of Department of Pathology, Bangladesh Agricultural University, Mymensingh and DR.
Md. Zubaed Hossain, MS student, Department of Physiology, Bangladesh Agricultural University,
Mymensingh for their inspirations and assistances during the course of the research.
The author would like to express his cheerful acknowledgements to the sweet surroundings of well
wisher specially DR. Md. Sydur Rahman Shawkat, DR. Md. Akhter Hamid, DR. Mahmud, DR.
Kabir, DR. Sankar, DR. Mohammad Ali, DR. Mehedi, DR. Mamun, DR. Harun, DR. Sujon, DR.
Suma and DR. Joyeta for their kind cooperation throughout the whole research period.
The author expresses his thanks to all technicians and staff especially Md. Idris Ali, Md. Raihan,
Department of Pathology and Md. Jalal Uddin, Department of Parasitology for their assistance.
The author gratefully acknowledges to his wife Mrs. Sabiha Khatun, elder brother M. Saiful Islam
and sister-in-law Mrs. Nilufar Yasmin for their marvelous sacrifices, inspiration and blessing
throughout his life.
Finally indebtedness is due to his beloved father late Abdullah and mother Samsun Nahar Begum for
their sacrifices, inspiration, cooperation and blessing to get him to this position.
The Author
November, 2011
ABSTRACT
Coccidiosis is a fatal disease of poultry. In order to combat caecal coccidiosis of poultry this
study was designed to evaluate the therapeutic and prophylactic efficacy of Toltrazuril at
different doses as well as its combination with other therapeutic and immunomodulatory
agents. Forty five, day old chicks, were randomized and divided into nine groups. Group A
served as noninfected, non-medicated control. The chicks of Group B, C, D, E, F, G, H and I
were challenged orally with 1x104 sporulated oocysts of E. tenella on the 10th and 15th day of
their age to induce infection. Chicks of Group B were infected and non-medicated while
chicks of Group C, D, and E were treated with Toltrazuril at a dose rate of 3.5, 7, and 12
mg/kg body weight respectively on day 22 and 23 of age; the early stage of clinical
coccidiosis. Chicks of Group F were treated on day 22 and 23 with Toltrazuril and Vitamin
K at a dose rate of 7mg/kg and 2.5 mg/kg body weight respectively. Similarly chicks of
Group G were treated with Toltrazuril 7 mg/kg and Sulfaclozine 150 mg/kg body weight and
the chicks of Group H were treated with Toltrazuril 7 mg/kg and Oyster Mushroom powder
100 mg/kg body weight. In chicks of Group I, Toltrazuril was given as prophylactic at a dose
rate of 3.5 mg/kg body weight from day 10 up to day 24 of experiment. The efficacy of
Toltrazuril was evaluated based on oocysts counts per gram (OPG) of faeces, weight gain,
morbidity, mortality and necropsy findings. The maximum reduction of OPG counts in
therapeutic trial on day 24 were detected in Group F (0.09±0.03 thousand, p<0.05) and
Group G (1.04±0.04 thousand, p<0.01). Concerning the mean body weight recorded as
compared to healthy control on day 24, Group F (850.75±2.40 g, p<0.01) chickens showed
highest performance in terms of therapeutic trial, followed by Group H (809.90±1.91 g,
p<0.01) and Group G (797.22±1.20 g, p<0.01) chickens. Chickens belonging to Group B
showed lowest performance in terms of body weight (643.97±1.649g) and OPG of faeces
count (64.44±1.38 thousand) (p<0.01). In prophylactic trial chickens of Group I showed the
highest performance in terms of body weight (942.60±1.44g) and OPG of faeces count
(0.03±0.01 thousand) (p<0.01). Results showed Toltrazuril as an effective prophylactic agent
against caecal coccidiosis of chicken and supplementation of Vitamin K in feed of infected
flock reduced morbidity and mortality.
vi
CONTENTS
CHAPTER TITLE PAGE ACKNOWLEDGEMENT iii-iv
ABSTRACT v
LIST OF CONTENTS vi-vii
LIST OF TABLES viii
LIST OF FIGURE ix
LIST OF ABBREVIATION AND SYMBOLS x
CHAPTER I INTRODUCTION 1-2
CHAPTER II REVIEW OF LITERATURE 3-13
2.1. Reviews on importance and prevalence in Bangladesh
3-4
2.2. Reviews on therapeutic and prophylactic agent 4-12
2.2.1. Reviews on drugs and drugs resistance 4-11
2.2.2. Reviews on mushroom 11
2.2.3. Reviews on Vitamin K 11-12
2.3. Reviews on pathogenecity of E. Tenella 12-13
CHAPTER III MATERIALS AND METHODS 14-27
3.1. Preparation of experimental house 14
3.2. Chicks 154
3.3. Feed 14-15
3.4. Collection of therapeutic agents 16
3.5. Management of the experimental chicks 16-17
3.5.1. Brooding 16
3.5.2. Feeding and drinking 16
3.5.3. Parasite 17
3.6. Production of fresh oocysts for experimental infection
17
3.6.1. Cleaning of oocysts 17
3.7. Experimental design 17-18
3.7.1. Groupings 17-18
3.7.2. Preparation of oocyst dose 18
3.7.3. Preparation of therapeutic and prophylactic doses
21
vii
CONTENTS (Contd.)
CHAPTER TITLE PAGE
3.7.4. Recording of daily weight 21
3.7.5. Recording of recovery and mortality rate 22
3.7.6. Counting of faecal oocyst 22
3.8. Post-mortem examination of broilers 22
3.9. Histopathological examination 22-27
3.9.1. Collection of samples 22
3.9.2. Preparation 10% buffered formalin 22-23
3.9.3. Chemicals required 23
3.9.4. Histopathological examination procedure 23
3.9.5. Processing of tissues 23-24
3.9.6. Staining procedure 24
3.9.6.1. Preparation of Harris hematoxylin solution 24
3.9.6.2. Preparation of eosin solution (1% stock alcoholic eosin)
24-25
3.9.6.3. Routine hematoxylin & eosin staining procedure
26-27
3.9.6.4. Histopathological studies & photomicrograph 27
3.10. Statistical analysis 27
CHAPTER IV RESULTS 28-41
4.1. Mean body weight record 28
4.2. OPG counts 28
4.3. Mortality and recovery rate record 28-29
4.4. Post-mortem findings after treatment 29
4.5. Histopathology 35-36
CHAPTER V DISCUSSION 42-45
CHAPTER VI SUMMARY AND CONCLUSION 46-47
REFERENCES 48-63
viii
LIST OF TABLES
TABLE TITLE PAGE
Table 1 Composition of the starter feed as provided by the manufacturer
15
Table 2 Composition of the grower feed as provided by the manufacturer
15
Table 3 Therapeutic agents used to study therapeutic and prophylactic potentials of caecal coccidiosis
16
Table 4 Doses for the therapeutic and prophylactic trial 21
Table 5 Preparation of 10% buffered formalin 22
Table 6 Time required for dehydrating tissues 23
Table 7 Preparation of Harris hematoxylin solution 24
Table 8 Preparation of eosin solution (1% stock alcoholic eosin) 24
Table 9 Therapeutic and prophylactic efficacy trial of Toltrazuril in broiler chicken in term of body weight gain
30
Table 10 Therapeutic and prophylactic efficacy trial of Toltrazuril in broiler chicken in term of OPG count
31
ix
LIST OF FIGURES
FIGURE TITLE PAGE Fig. 1
Different therapeutic agents used for the management of induced caecal coccidiosis in chickens
19
Fig. 2 Vaccination of chicks with BCRDV on day 2 19 Fig. 3 Sporulated oocyst of Eimeria tenella (arrow) derived after culture. 20 Fig. 4 Feeding in the experimental Group B on day 11 20 Fig. 5 Post mortem examination of the chickens of Group A did not reveal
any major lesion 32
Fig. 6 Distended caeca filled with blood tinged contents as seen in Group B 32 Fig. 7 Profuse haemorrhage was seen after opening up the caecal tube of
Group B chicken that died of induced coccidiosis 33
Fig. 8 Haemorrhagic lesion was seen in the mucosa after washing up the caeca of Group D chicken
33
Fig. 9 Caeca after washing of Group F showing no lesion on day 24 34 Fig. 10 Caecal section of the chicken of Group A (healthy control) did not
reveal any significant microscopic lesion (H & E, 100X) 37
Fig. 11 Caecal section of the chicken of Group B (infected untreated) showing desquamation of epithelia, merozoites/schizonts in mucosa, and infiltration of reactive cells (H & E, 100X)
37
Fig. 12 Caecal section of chicken of Group C treated with 3.5 mg Toltrazuril /kg body weight showing erosion and desquamation of crypt epithelia, infiltration of reactive cells in the lamina propria with merozoites/schizonts in the crypt of villi (H & E, 400X)
38
Fig. 13 Caecal section of chicken of Group D treated with 7 mg Toltrazuril /kg body weight showing arrested coccidial development with necrosis, haemorrhages, erosion, and desquamation of epithelial cells (H & E, 100X)
38
Fig. 14 Caecal section of chicken of Group E treated with 12 mg Toltrazuril /kg body weight showing haemorrages in caecal mucosa and submucosa, infiltration of inflammatory cells (H & E, 100X)
39
Fig. 15 Caecal section of chicken of Group F treated with7 mg Toltrazuril and 2.5 mg Vitamin K/kg body weight showing diffuse infiltration of reactive cells specially eosinophils and mononuclear cells in the mucosa and submucosa, degeneration of caecal gland but no significant haemorrhage present (H & E, 100X)
39
Fig. 16 Caecal section of chicken of Group G treated with7 mg Toltrazuril and 150 mg Sulfaclozine/kg body weight showing slight desquamation of caecal epithelia, necrosis, and degeneration of mucosa and caecal gland (H & E, 100X)
40
Fig. 17 Caecal section of chicken of Group H treated with7 mg Toltrazuril and 100 mg Oyster mushroom/kg body weight showing desquamation of epithelial lining, infiltration of reactive cells in mucosa and submucosa (H & E, 100X)
40
Fig. 18 Caecal section of chicken of Group I (used as prophylactic trial) treated with3.5 mg Toltrazuril/kg body weight showing no significant lesion related to coccidiosis (H & E, 100X)
41
x
LIST OF ABBREVIATION AND SYMBOLS
BAU : Bangladesh Agricultural University
BCRDV : Baby chick Ranikhet disease vaccine
cm : Centimeter
Co. : Company
dl : Deciliter
et al. : Associate
etc. : Etcetera
Fig. : Figure
Kcal Kilocalorie
kg : Kilogram
g : Gram
Ltd. : Limited
Max. : Maximum
Min. : Minimum
No. : Number
OPG : Oocyst per gram
rpm : Rotation per minute
SE : Standard error
µm : Micrometer
ºC : Degree Celsius
± : Plus minus
CHAPTER I
INTRODUCTION
CHAPTER I
INTRODUCTION
Poultry farming in Bangladesh has grown as an emerging and prospective industry and many
landless farmers are found to involve with poultry rearing (Huque, 2001). A total of 5 million
people are engaged in this sector (Saleque, 2006). At present chicken contributes 51% of
total meat production in Bangladesh (Raha, 2007). There are about 110,800 small and large
scale poultry farms in this country (Anon, 2006) and per capita annual consumption of meat
is 5.99 kg against the universal standard 80 kg per head (Raha, 2007).
There are several constraints of poultry industries in Bangladesh including outbreak of
infectious diseases causing economic loss and discouraging poultry rearing (Das et al.,
2005). Among the different diseases, parasitic infection brings a great threat to poultry
industry. Coccidiosis is a common and fatal disease in poultry. Intestinal coccidiosis, caused
by various species of Eimeria, is an economically important (estimated to be 2 billion dollars
a year) disease of poultry (Zhang and Zeng, 2005). Eimeria spp. are belonging to the phylum
Apicomplexa causing coccidiosis of farm animals and birds. Eimeria tenella is the most
important species, as it causes caecal coccidiosis in chickens (Shirley, 1986). Eimeria tenella
primarily invades and resides in the linings of caeca of exposed chickens (Vervelde and
Vermeulen, 1995 and Yun et al., 2000).
In Bangladesh a number of drugs are available for the treatment and prevention of
coccidiosis. Anticoccidial drugs remain important for a long time, although resistance
development could limit their use (Stephen et al., 1997). Moreover, the price of synthetic
anticoccidials is too much high and efficacy is not so satisfactory. Anticoccidial therapeutic
agents must fulfill some main criteria including a high level of efficacy against all
developmental stages of pathogenic Eimeria species infecting poultry and at the same time,
they shouldn't interfere with the immune response of the host during and after treatment of
coccidial infections at therapeutic dosages (El-Banna et al., 2005).
Toltrazuril is a symmetrical triazinetrione compound and 2.5% oral solution has been shown
to be effective against all species of Eimeria infecting chickens (Mehlhorn et al., 1988). It is
active against all intracellular developmental stages including those of schizogony and
gametogony (Mehlhorn et al., 1984). Toltrazuril has chemoprophylactic (Gjerde and Helle
1991) and therapeutic effects (Chapman, 1987; Mehlhorn et al., 1988; Mathis et al., 2004;
Ghanem et al., 2008) against coccidiosis and does not interfere with the development of
immunity (Grief, 2000). Chemoprophylaxis with Toltrazuril enhances immunity against
poultry coccidiosis (Grief, 2000). It has been proved that therapeutic medication with
Toltrazuril protects the birds from clinical coccidiosis (Ramadan et al., 1997).
There are other different therapeutic and prophylactic agents for coccidiosis in different
species. Among these vitamin-K added to a deficient diet reduces mortality due to Eimeria
tenella or E. necatrix (Ryley et al., 1978), sulfaclozine may reduce the deleterious effects of
coccidiosis in broilers (Sanei et al., 2009) and dietary supplementation with oyster
mushroom powder enhances anti-inflammatory activity which is mediated through the
inhibition of NF-κB and AP-1 signaling (Jedinak et al., 2011).
To evaluate therapeutic and prophylactic potentials of Toltrazuril against poultry coccidiosis
this study was undertaken with the following objectives:
i. To study the therapeutic potentials of Toltrazuril against caecal coccidiosis in
chickens.
ii. To study the prophylactic effect of Toltrazuril against caecal coccidiosis in
chickens.
iii. To compare the therapeutic and prophylactic potentials of Toltrazuril against caecal
coccidiosis in chickens in terms of body weight gain, oocyst counts per gram (OPG)
of faeces, morbidity and mortality pattern and postmortem lesions.
CHAPTER II
REVIEW OF LITERATURE
3
CHAPTER II
REVIEW OF LITERATURE
It is true that coccidiosis is one of the most serious problems in poultry industry
throughout the world including Bangladesh. A few relevant published information on the
experiment have been reviewed in the following paragraphs. The reviews have been
arranged in the chronological order.
2.1. Reviews on importance and prevalence in Bangladesh
Coccidiosis in poultry appeared as an important disease as it is caused higher mortality
and morbidity in farm practices.
Kutubuddin (1973) made a study on mortality due to coccidiosis of chicken in BAU
poultry farm and recorded 14.66% mortality of the birds due to coccidiosis. Sarker
(1976) collected 300 dead birds from Bangladesh Agricultural University (BAU) Poultry
farm and based on post mortem examination about 12% infectivity with coccidiosis was
recorded.
Mondal and Qadir (1978) reported subclinical coccidiosis (54.14%) of chicks in (BAU)
poultry farms and among the infected birds 23.75% were found to infect with E. tenella,
the most predominant species in Bangladesh. Karim (1988) studied chicken coccidiosis
in Bangladesh, and reported 24.8% of mortality of chicken due to coccidiosis in October,
1986.
Karim and Tress (1990) identified coccidiosis as one of the major diseases of poultry in
Bangladesh. The authors also identified five species of Eimeria named E. tenella, E.
necatrix, E. acervulina, E. maxima and E. brunetti, that cause coccidiosis of chicken in
Bangladesh. Karim and Begum (1994) reported the incidence of E. tenella as a major
species of chicken coccidia caused great economic loss to the poultry industry in
Bangladesh. The authors also reported two additional species: E. precox and E. mitis in
chicken with high morbidity and mortality.
4
Karim et al (1994) recorded higher prevalence of chicken coccidiosis during winter or
cold months, and found E. tenella (70%), E. maxima (40%), E. brunetti (30%) and E.
necatrix (20%) by lesion scoring of birds. The authors reported that E. acervulina was the
commonest species and found in 80% birds examined.
Bhattacharjee et al (1996) made a study on prevalence of chicken coccidiosis in
Bangladesh and reported the prevalence is 9.4% cases. Islam et al. (1996) investigated
the prevalence of chicken coccidiosis in dead birds. The authors found that 39.2% of the
birds (out of 337) were affected with coccidiosis.
Talha (1999) studied the prevalence of coccidiosis in different poultry farms of
Bangladesh. The author reported 45% mortality rate due to chicken coccidiosis in
Bangladesh. Karim (2001) reported the second most pathogenic species is E. necatrix but
least common in Bangladesh.
Giasuddin et al. (2002) did a statistics on mortality due to coccidiosis in Bangladesh and
reported 4-5% mortality rate in chicken due to coccidiosis in Bangladesh. Giasuddin et
al. (2003) made a study on the prevalence of coccidiosis in chicken in Bangladesh and
reported 9.17% prevalence.
Saleque (2003) collected data (1999-2002) from Central Disease Investigation
Laboratory and BRAC laboratory, and reported gradual higher prevalence of coccidiosis
in poultry farms over the last 3 years.
2.2. Reviews on therapeutic and prophylactic agents
2.2.1 Reviews on drugs and drug resistance
Warren et al. (1966) examined the response of field strains of coccidia to several
coccidiostats during 1964 and 1965. The authors reported strains of all species of
Eimeria, and more strains of coccidia had a tendency to be resistant to coccidiostats that
were used. The coccidiostats used included 0.0125% amprolium, 0.009% coccidiostat `D'
(a mixture of 8 part sulphaquinoxaline and 1 part diaveridine), 0.01% nitrofurazone,
0.0145% coccidiostat `P' (a mixture of 16 percent amprolium, 12 parts sulphaquinoxaline
and 1 part ethopabate), 0.0125 percent sulphaquinoxaline and 0.125 percent zoalene.
5
Joyner (1970) in a study reported resistant response to coccidiostatic drugs in different
strains of Eimeria spp. The author also reported that the drug resistance may be induced
to most coccidiostat in common use and failure of prophylaxis may occur in the presence
of resistant strains.
Oikawa et al.(1975) reported that 97% of the strain of E. acervullina was resistant to
decoquinate and 93% resistant to clopidol; a considerable percentage of the strain of E.
tenella and E. necatrix were resistant to amprolium, clopidol and decoquinate, and only a
small percentage of these strains were resistant to sulfadimethoxime.
Chapman (1978) studied on the development of resistance in the Houghton strain of E.
tenella to anticoccidial drugs like amprolium, clopidol and methyl benzoquate. The
author reported that amprolium and clopidol developed resistance more rapidly in
experiments.
Gill and Bajwa (1979) studied on drug resistance of different field isolates of Eimeria
spp. The authors reported 71.5% resistant to sulphaquinoxaline, 58.8% to bifuran, 34.1%
to amprolium, 6.3% to clopidol and 5.6% to nicarbazin against chicken coccidiosis.
Jeffers and Bentley (1980) reported that polyether anticoccidials as an ineffective
coccidiostat against Eimeria spp and developed resistance against coccidia readily.
Krylov and Zaionts (1981) reported that 97.5% field isolates of E. tenella exhibited
resistance to nitrofuran derivatives, 75% to 3, 5-dinitro denzamide derivatives, 47.5% to
sulphanylantides and 10% to thiamin derivatives.
Mathis and McDougald (1982) studied the efficacy of clopidol, amprolium, nequinate,
zoalene and sulfaquinoxalines, and found that E. tenella, E. acervulina and E. maxima
had high frequency of resistance against these drugs. On the other hand, very few isolates
were resistant to nicarbazin, robenidine or halofuginone.
Chapman (1982) collected field isolates of E. acervulina from broiler and breeder farms
throughout UK and reported sulphaquinoxaline and suophaquinoxaline plus
pyrimethamine were only partially effective coccidiostat against the field isolates.
6
Chapman (1982) studied over a period of one year in UK and collected isolates from
broiler farms where monensin had been used continuously for 28 and 38 crops (broiler
isolates) and from breeder farms where the drug had never been used (breeder isolate).
The author reported that the broiler isolates were partial resistant to monensin.
Chapman (1983) reported that several Eimeria spp. were resistant to aprinocid which
had been used for 5 to 7 successive flocks @ 60 ppm in diet. Few isolates were found
sensitive where aprinocid had never been used.
Chapman (1984) studied with Houghton strain of E. tenella and found that after 16
passages the parasite become resistant (partial) to monenisn at the concentration of 100
ppm. At higher concentrations of the drug, the parasite was not resistant. Chapman
(1986) studied the resistance of monensin against chicken coccididosis and reported that
the field isolates of E. tenella were partly resistant to monensin.
Sanda (1986) made a therapeutic trial to study the efficacy of monensin against chicken
coccidiosis. The author supplied monensin in feed for 21 days in E. tenella infected
chicks and the birds were recovered well.
Rommel (1987) observing the resistant strains of Eimeria, and stated that it was
necessary to seek an alternative to the use of anticoecidials because of the development of
resistant coccidial parasite and need to reduce drug residues in tissues.
Salisch (1987) used maduramicin at 6 ppm and chicken coccidiosis was controlled.
Maduramicin treatment resulted in significantly lower lesion scores and fewer parasites
(oocysts) were seen in litter samples.
Munoz and Rodriguez (1988) used maduramincin, monensin, salinomycin, narasin and
Iasalocid in the treatment of Eimeria spp. The authors observed highest Anticoccidial
Index (AI) activity in maduramicin treated group at different dose rates (AI = 164.59,
169.37 and 170.24 at 5 ppm, 6 ppm and 7 ppm respectively).
Varga et al. (1988) studied anticoccidial activity of maduramicin, monensin, narasin,
salinomycin and lasalocid. The authors used maduramicin ammonium at the
recommended dietary level of Smg/kg body weight in battery and floor pen trials. In both
7
the trials, the authors found the efficacy of maduramicin superior to other ionophorous
anticoccidials against E. tenella and E. accrvulina infection.
Reather and Paeffgen (1989) studied the sensitivity to monensin (100 ppm); narasin (70
ppm), salinomycin (60 ppm), maduramicin (5 ppm) and lasalocid (90 ppm) of coccidiosis
infected broiler chickens in nine European countries. The authors reported that five of 26
isolates were sensitive to- all ionophorous drugs tested, while the rest of the isolates
(81%) were partially or entirely resistant to one or several polyether. E. tenella and E.
brunetti isolates were highly resistant to ionophores.
Harder et al. (1989) tested that the anticoccidial properties of toltrazuril in Eimeria
falciformis-infected mice and were potentiated by the simultaneous application of
pyrimethamine, trimethoprim, or sulfadimidine. Their results suggest that toltrazuril
primarily affects the respiratory chain and secondarily, two enzymes involved in
pyrimidine synthesis.
Salisch and Shaksholik (1989) studied the efficacy of ionophorous drugs against E.
tenella and E. acervulina. The authors reported that salinomicin and maduramicin were
most effective against E. acervulina and E. tenella respectively. On the other hand,
maduramicin was least effective against E. acervulina and monensin was slightly
effective against E. tenella.
Salisch and Friederichs (1991) studied the efficacy of maduramicin and salinomycin
against E. brunetti. The authors reported that maduramicin (5 ppm in feed) gave better
result by suppressing the oocysts production completely.
AL-Taee et al. (1993) studied the efficacy of maduramicin and monensin against E.
tenella infection in chicken. The authors reported that maduaramicin is more effective
than monensin.
Zhang et al. (1995) studied the efficacy of maduramicin against E. tenella infection in
chicken. The authors reported that the recommended dose of maduramicin was 2.5-4 ppm
for prevention and 4-5 ppm for treatment against E. tenella infection.
8
Grief et al. (1996) reported that 55% strains from 15 E. acervulina and five E. brunetti
strains were resistant to several anticoccidial drugs like maduramicin (5 ppm), monensin
(100 ppm), salinomysin (6fl ppm), nicarbazin (125 ppm), halofnginone (3 ppm),
diclazuril (1 ppm) and toltrazuril (25 Ppm).
Stephan et al. (1997) studied ten Eimeria field isolates from North Germany in battery
tests for sensitivity to selected anticoccidials. Chapman (1997) studied that anticoccidial
drugs are widely used for the control of coccidiosis in the fowl which has inevitably led to
the development of drug resistance.
Greif (2000) studied immunoglobin level and activity of immune sera after toltrazuril
treatment; toltrazuril may be involved in the enhancement of immunity. Chapman (2000)
studied that immunological response is recognized as the only major practical alternative
to chemotherapy for the control of coccidiosis.
Lakkundi et al. (2002) studied the effect of toltrazuril and amprolium on body weight
and feed efficiency of broiler chicken experimentally infected with Einreria tenella. The
authors reported that toltrazuril (25 ppm) was very effective irrespective of degree of
infection, whereas the efficacy of amprolium (240 ppm) was better in moderate than in
heavy infection.
Lakkundi et al. (2002) evaluated the effect of toltrazuril and amprolium on body weight
and feed efficacy in broiler chickens experimentally infected with caecal coccidiosis.The
authors found that Toltrazuril treated birds did not excrete oocysts, but had better body
weight gain and feed efficiency than uninfected birds. The caecal lesion score was mild in
this group. In the amprolium treated birds, the OPG was less than in infected controls and
mean body weight was lower than toltrazuril treated birds. However, cumulative feed
efficiency was similar in both treatments. The lesion score was moderate in amprolium
treated birds. The results indicated that toltrazuril was very effective irrespective of
degree of infection, whereas the efficacy of amprolium was better in moderate than in
heavy infection.
Lakkundi et al. (2002) evaluated histopathological anticoccidial activity of toltrazuril
and amprolium in experimentally induced caecal coccidiosis in broiler chicken (n=280). It
9
was reported that toltrazuril prevented the establishment of caecal coccidiosis by
degeneration and disintegration of the first generation of schizonts. However, in the
amprolium-treated birds, few intact first and second generation schizonts and also micro
and macrogametocyte s were noticed on microscopic examination. Occasionally, oocysts
were also found. It was concluded that toltrazuril has a coccidiocidal effect, whereas
amprolium is coccidiostatic in nature based on the histopathological studies.
Jin-GuangMing et al. (2003) selected one hundred and thirty 21-day-old healthy
chickens to study the resistance of two Eimeria tenella isolates, Nanjing isolate and
Fengyang isolate, to diclazuril, maduramicin, clopidol, robenidine and amprolium. The
authors reported that two isolates were light resistant to amprolium, sensitive or light
resistant to diclazuril, middle resistant to clopidol, middle resistant or complete resistant
to maduramicin and robenidine.
Ghazala-Nawaz et al. (2003) tested the efficacy of 2.5% toltrazuril against
experimentally induced coccidiosis in broiler chicks. It was shown that treatment with
2.5% toltrazuril at 7 mg/kg in drinking water for 2 consecutive days, for 8 h per day was
effective for controlling experimentally induced acute coccidiosis and immediately
stopped mortality.
Grilli et al. (2003) conducted a study to investigate the effect of a therapeutic medication
with toltrazuril on the control of coccidiosis and broiler performance in the absence of
coccidiostat-medicated feed. They reported that the toltrazuril single treatment controlled
coccidiosis and oocyst shedding was decreased at 35 days of age. The results indicated
that in case of early coccidiosis challenge, the treatment should be anticipated between
10-14 days of age to avoid death of chicken due to damage at earlier onset.
Mathis et al. (2003) conducted a 42-day broiler floor pen study conducted comparing the
anticoccidial efficacy of toltrazuril (Baycox) as a stand alone treatment and as an
additional treatment to in-feed anticoccidial programs. The authors reported that
Toltrazuril most completely eliminated all coccidial lesions and dramatically reduced
oocyst shedding and Toltrazuril can thus be used for supplemental control with in-feed
anticoccidials or as a primary anticoccidial with nonmedicated feed.
10
Dhillon et al. (2004) tested the efficacy of toltrazuril against 6 different levels of Eimeria
tenella infection in 2-week-old chicks was evaluated. The authors reported that the
treatment resulted to lower mortality and reduced oocyst production. Moreover, therapy
resulted to complete elimination of the clinical signs at lower levels of infection. The drug
interfered in the development of immunity only at the lower levels of infection.
Mathis et al. (2004) conducted a 56-day floor pen study to determine the appropriate
time to administer toltrazuril (BaycoxReg.) for control of coccidiosis in broiler chickens.
They reported that the final performance for the salinomycin (SAL/SAL) was
significantly less compared to all toltrazuril and nicarb / salinomycin (NIC/SAL) birds.
All toltrazuril treatments at days 2-3 provided good coccidiosis control with
accompanying performance. The absence of clinical coccidiosis relapse during the last
third of the growout along with moderate oocyst counts and low lesions was indicative of
unimpaired coccidiosis immunity.
Sheng-chao et al. (2005) studied efficacy evaluation of combinations among selected
anticoccidials against Eimeria tenella in chicken. Results showed that the combination of
polyether ionopgorous antibiotics and synthetic chemical coccidials acted later in the life
cycle of E. tenella had the synergistic efficacy against coccidiosis and the good promotion
of weight gain in chickens.
Claeskens et al. (2007) assessed Toltrazuril (BaycoxReg.) stand-alone treatment and
Paracox-5 (live attenuated vaccine) in a field trial for broiler coccidiosis control. They
found that within treatment groups, the feed conversion ratio and daily weight gain were
significantly influenced by the month of hatch. It was concluded that a single treatment
with toltrazuril is a valuable alternative to vaccination for coccidiosis control in a rotation
programme.
Sanei et al. (2009) studied the effect of sulfaclozine 30% (Esb3) on experimental
coccidiosis in broiler cockerels. They reported that sulfaclozine may reduce the
deleterious effects of coccidiosis in broilers. Sentepe and Eraslan (2010) studied the
Pharmacokinetic of sulfaclozine in broiler chickens.
2.2.2. Reviews on mushroom
11
Manzi et al. (1999) made a comparative study on various components of nutritional
interest, such as water, protein, total amino acids, ash and minerals, in mushrooms of
different species (Pleurotus ostreatus, Pleurotus eryngii, Pleurotus pulmunarius and
Lentinula edodes).
Wasser (2002) made a study on medicinal mushrooms. The author reported that these
mushroom represent an unlimited source of polysaccharides with antitumor and
immunostimulating properties.
Borchers et al. (2008) studied the immunobiology of mushroom. They reported that
mushrooms have beneficial effects on immune function with subsequent implications for
inhibition of tumor growth.
Jedinak et al., 2011 done a study on anti-inflammatory activity of edible oyster
mushroom. They reported that oyster mushroom (Pleurotus ostreatus) has significant
anti-inflammatory effect which is mediated through the inhibition of NF-κB and AP-1
signaling.
2.2.3. Reviews on Vitamin K
Dam (1942) studied the chemistry and physiology of Vitamin K. Stephens et al. (1960)
performed research on sources and levels of vitamin k in relation to cecal coccidiosis.
They reported that Vitamin K supplementation reduces the mortality of chicken due to
caecal coccidiosis.
Harms et al. (1960) made a comparison of various sources and levels of vitamin k
activity using chicks with cecal coccidiosis. Warren (1968) studied the Vitamin
requirements of the Coccidia of the chicken. He reported that for normal development of
the parasites, Eimeria acervulina and E. tenella in the chicken, dietary thiamine,
riboflavin, biotin, nicotinic acid and folic acid are required. E. acervulina required
thiamine for second schizogony and sporulation; riboflavin for first schizogony and
possibly gametogony, biotin for the development of the sporozoites/trophozoites and for
gametogony, and nicotinic acid for schizogony and gametogony. E. tenella required
12
thiamine and riboflavin for gametogony; biotin for first schizogony; and nicotinic acid for
second schizogony.
Ryley et al. (1978) studied the use of vitamin k deficient diets in the screening and
evaluation of anticoccidial drugs. They found that Vitamin K (as menaphthone sodium
bisulphite) added to a deficient diet reduced mortality due to Eimeria tenella or E.
necatrix, had a slight effect on haematocrit, but had no obvious effect on weight gain or
faecal blood; 0·1 ppm gave a maximal response. The effect of vitamin K on mortality was
not absolute; the magnitude of the effect depended on the size of the challenge dose of
oocysts.
2.3. Reviews on pathogenecity of E. tenella
Tyzzer (1929) reported that E. tenella was the most pathogenic species of all the avian
coccidia. Waxlen (1941) found that death occurred due to haemorrhage associated with
second generation sehizonts of E. tenella.
Goodrich (1944) observed that oocysts of E. tetella survived for 24 to 25 months and
retained its patlaogeneciy at room temperature in 2.5% potassium dichromate solution.
Edgar (1955) reported that the prepatent period of several species of coccidia were one
day less than that was reported previously. The author also stated that sporulation time of
E. tenella was only 19 hours at 29°C, 21 hours at 26°C and 24 hours at 20°C.
Doran and Vetterling (1969) reported 23-59% mortality in 3 week old chicks infected
with 105 oocysts of E. tenella. Long (1973) studied the pathogenecity of E. tenella and
reported that there was haemorrhage, sloughing of caecal mucosa, thinning of caecal wall
and eventual fibrosis and thickening of the walls.
Dikovskaya (1974) studied the pathogenecity of E. tenella in different areas of USSR and
recorded 12.5-80% mortality of chicks by this parasite. Mondal and Qadir (1978)
identified 23.75% infection in chicken due to E. tenella which is the commonest one.
Seeripto (1984) observed that infection of village chickens with 5 x 104 oocysts of E.
tenella cause only a slight decrease in body weight, but the layers and broilers showed
clinical signs of coccidiosis and had a weight loss of 19.4% and 14.2% respectively.
13
Mortality was recorded in village chickens when the inoculated dose of oocysts was
increased from 5 x 104 to 15 x 104, but the rate was much lower than in broilers.
Nayak and Rai (1985) studied the pathogenecity of several species of Eimeria in broiler
chickens. The authors reared day old broiler chicks in presterilized experimental cages in
similar environmental conditions, food and water. At the age of 6 weeks the birds were
infected orally with 2 x 104 oocysts of E. tenella, E. necatrix and E. acervulixa in mixed
culture. The authors reported that the total erythrocyte count, haemoglobin and packed
cell volume (PCV) were significantly lower in infected birds.
Shirley and Millard (1986) studied the pathogenecity of E. tenella in chickens. The
authors gave treatment of several infected birds, but unfortunately the authors found no
difference in body weight gain of treated and untreated birds.
Jungmann and Mielke (1989) studied on E. tenella irradiated vaccine and reported that
the immunized chickens challenged with E. tenella developed no clinical sign. On the
other hand, the controls (E. tenella infected, not immunized) had severe haemorrhagic
enteritis with 88% mortality.
CHAPTER III
MATERIALS AND METHODS
14
CHAPTER III
MATERIALS AND METHODS
The experiment was conducted at the Department of Pathology, Faculty of Veterinary
Science, Bangladesh Agricultural University (BAU), Mymensingh to study the
therapeutic and prophylactic potentials of Toltrazuril against caecal coccidiosis of
chicken.
3.1. Preparation of experimental house
The experimental poultry house was properly cleaned, washed and then dried up. The
room was fumigated with formaldehyde and with ammonia before introduction of
chicks. The feeder, waterer and cages were cleaned with water and then fumigated
with ammonia.
3.2. Chicks
Forty five, Cobb 500 day old chicks were included in this study. The chicks were
collected from Kazi Hatchery, Gazipur. Vaccination of chicks with BCRDV was
performed on day 2 and 19 (Fig.2).
3.3. Feed
The broiler chicks were provided with standard broiler starter and grower ration (Champion
Starter and Champion Grower, Quality Feeds Ltd.) according to the age (Table 1 and 2). The
starter feed was given from day1 to day 10 and the grower feed was given from day 11
up to the end of the experiment. The feed was stored in a dry and coccidia free area.
15
Table 1: Composition of the starter feed as provided by the manufacturer
Sl. No. Feed composition Percentage (%)
1. Humidity (max.) 11
2. Protein (min.) 24.5
3. Fat (min.) 5
4. Carbohydrate (max.) 4
5. Lysin (min.) 1.4
6. Methionine (min.) 0.65
7. Calcium (min.) 1.2
8. Phosphorus (min.) 0.75
Total metabolic energy 3150 kcal/kg
Table 2: Composition of the grower feed as provided by the manufacturer
Sl. No. Feed composition Percentage (%)
1. Humidity (max.) 11
2. Protein (min.) 24
3. Fat (min.) 5
4. Carbohydrate (max.) 4
5. Lysin (min.) 1.4
6. Methionine (min.) 0.60
7. Calcium (min.) 1.2
8. Phosphorus (min.) 0.75
Total metabolic energy 3175 kcal/kg
16
3.4. Collection of therapeutic agents
The therapeutic agents for the experiment (Fig.1) are presented in Table 3.
Table 3: Therapeutic agents used to study therapeutic and prophylactic potentials of
caecal coccidiosis
Sl. No. Therapeutic agent Trade name Source
1. Toltrazuril Cox-Zero ® Local market
2. Vitamin-K Rena-K® Local market
3. Sulfaclozine Nava Cox® Local market
4. Oyster mushroom
powder
- Horticulture Center, Kewatkhali,
Mymensingh, Bangladesh.
3.5. Management of the experimental chicks
The following procedures were adapted during the experimental period.
3.5.1. Brooding
The experimental chicks were brooded in two brooders provided with temperature of
35°C ± 1°C at 1st week of age and then gradually decreased the temperature at the rate
of 2.5°C per week up to 24 days of age. Temperature was corrected by adjusting the
height of the heat source, i.e. the electric bulb used for heating. The birds were reared
in coccidia free condition as the utensils were sprayed with ammonia earlier. Strict
biosecurity was maintained.
3.5.2. Feeding and drinking
The chicks were given feed twice a day in the morning and in the afternoon, and water
ad libitum. During the 1st week feed was supplied on large brown paper and water in
waterer. After 1st week the feeders were provided. Feeders and waterers were cleaned
daily. After 10 days when the chicks were placed in cages then water was given in
beakers.
17
3.5.3. Parasite
A single oocyst derived strain of E. tenella as a field isolate was used in this
experiment which was collected from a chicken affected with caecal coccidiosis.
3.6. Production of fresh oocysts for experimental infection
For the production of fresh oocysts, each seven days old chicks (n=6) was infected
with 104 sporulated oocysts. Faeces were collected from 10-11 days post infection
(dpi) and were kept in 2% potassium dichromate solution, while the concentration of
oocyst was found in higher concentration than oocysts (Fig.3) were collected and used
in this study.
3.6.1. Cleaning of oocysts
The oocysts were cleaned from the faeces by adopting the method described by Ryley
(1973) with required modifications. After collection in 2% potassium dichromate
solution the faeces was homogenized using a food blender. The homogenized mixture
was then centrifuged at 700 rpm for 5 minutes and the supernatant was discarded. The
sediment was resuspended by using saturated salt solution (specific gravity 1.200) and
centrifuged at 500 rpm for 5 minutes. With the help of a plastic Pasteur pipette the
oocyst rich scum was collected from the supernatant. The oocysts suspension was then
passed through a mash (90 µm apperture) sieve collected from local market. The
suspension was diluted with water to at least ten times of its original volume. After
centrifuging the suspension at 1100 rpm for 5 minutes the supernatant was discarded.
The oocysts rich sediment was resuspended in 2% potassium dichromate and aerated at
room temperature for 48 hours. The sporulation percentage was determined by
counting a total of 100 oocysts (both sporulated and unspolsulated). The oocysts were
stored at 4°C temperature for subsequent use.
3.7. Experimental design
3.7.1. Groupings
The chicks (n = 45) at the age of day 10 were divided into 9 equal groups (Group A, B,
C, D, E, F, G, H and I). The chicks were reared in separate cages. The Group A was
18
maintained as a control group without induction of infection and treatment and Group
B was maintained as infected but untreated (Fig.4). The next 6 groups (Group C to H)
were used for therapeutic trial (Table 4) and Group I was used for prophylactic trial
(Table 4) exposed to coccidial infection.
3.7.2. Preparation of oocyst dose
Oocyst suspension was centrifuged at 500 rpm for 5 minutes in test tubes and the
supernatant was discarded. The sediment after resuspending in distilled water was
centrifuged again. This process was repeated until the potassium dichromate was
cleaned off. The final sediment was resuspended in distilled water and the number of
oocysts per milliliter was counted by McMaster counting technique. Finally the
number of oocysts as infective inocula (104 sporulated oocysts) was adjusted to a
volume of 0.5 to 1 ml with water and each prepared dose was administered orally to
every chick of Group B, C, D, E, F, G, H and I on day 10 and 15.
19
Fig.1: Different therapeutic agents used for the management of induced caecal
coccidiosis in chickens.
Fig. 2: Vaccination of chicks with BCRDV on day 2.
20
Fig.3: Sporulated oocyst of Eimeria tenella (arrow) derived after culture.
Fig.4: Feeding in the experimental Group B on day 11.
21
3.7.3. Preparation of therapeutic and prophylactic doses
After appearance of clinical signs and symptoms the chicks of different groups were
treated as follows:
Table 4: Doses for the therapeutic and prophylactic trial
Grouping Days of
treatment
(from- to)
Treatment
Group A (Control group) - Without treatment
Group B (Infected untreated) - Without treatment
Group C 22-23 3.5 mg Toltrazuril/kg body weight
Group D 22-23 7 mg Toltrazuril/ kg body weight
Group E 22-23 12 mg Toltrazuril/ kg body weight
Group F 22-23 7 mg Toltrazuril and 2.5 mg
Vitamin K/ kg body weight
Group G 22-23 7 mg Toltrazuril and 150 mg
Sulfaclozine/ kg body weight
Group H 22-23 7 mg Toltrazuril and 100 mg
Oyster mushroom/ kg body weight
Group I (used in prophylactic
trial)
10-24 3.5 mg Toltrazuril/ kg body weight
3.7.4. Recording of daily weight
Chicks were weighed individually from the day of starting (day 1) till the end of the
experiment (day 24) every day at morning. The average mean individual weight was
calculated from these values.
22
3.7.5. Recording of recovery and mortality rate
In response to the treatment the recovery and mortality rate were recorded up to the
end of the experiment.
3.7.6. Counting of caecal oocyst
The faeces were collected every day after post infection immediately after defecation.
The faeces of all groups were homogenized in a food blender separately and made up
to 0.5 liter by adding water. After thorough mixing by gentle stirring, 1 ml of
suspension was diluted in the saturated salt solution to make a desired dilution (x 10)
and mixed by gentle shaking. A portion of the suspension was withdrawn with the help
of an ordinary plastic transfer pipette and the two chambers of McMaster egg counting
slide was filled. After 3-5 minutes the oocysts in the two chambers, were counted by
using low power objective (x 10). The number of oocysts per bird was calculated by
dividing the figure by 0.3 and multiplied by the dilution factor and then divided by the
number of chicks in each subgroup.
3.8. Post-mortem examination of broilers
At the end of experiment (day 24), post-mortem examination of the broilers were
performed. Caeca, intestine, liver and spleen were examination. The gross post-
mortem findings of these organs were recorded. The caeca were collected for
histopathology.
3.9. Histopathological examination
3.9.1. Collection of samples
Caeca from the experimental chickens were collected in 10% neutral buffered
formalin and used for histopathological study.
3.9.2. Preparation 10% buffered formalin
Table 5: Preparation of 10% buffered formalin
37-40% formalin 100 ml
Distilled water 900 ml
Sodium Phosphate (monobasic) 4 gm
Sodium Phosphate (dibasic) 6.5 gm
23
The above ingredients were mixed thoroughly, preserved in an air tight container
split in plastic jar @ 250ml/jar
3.9.3. Chemicals required
(i) Alcohol (50%, 70%, 80%, 95% & absolute)
(ii) Chloroform
(iii) Paraffin
(iv) Xylene
(v) Distilled water
(vi) Hematoxylin
(vii) Acid alcohol
(viii) Ammonium water
(ix) Eosin
3.9.4. Histopathological examination procedure
Fixed tissue sections (caeca) were processed for paraffin-embedding, sectioning
(Luna, 1968) and staining with Hematoxylin & Eosin stain.
3.9.5. Processing of tissues
The formalin fixed tissues were properly trimmed. The tissues were washed
overnight under running tap water to remove formalin. The tissues were
dehydrated in ascending grades of alcohol.
Table 6: Time required for dehydrating tissues
50% alcohol 1 hour
70% alcohol 1 hour
80% alcohol 1 hour
95% alcohol 1 hour
100% alcohol 1 hour X 2
The tissues were cleared in two changes in chloroform, 1.5 hour in each. The
tissues were embedded with molten paraffin wax at 560C for two changes, 1.5
hour in each. Paraffin block containing tissue pieces were made using templates.
The tissues were sectioned with a microtome at 5 µm thickness, allowed to
spread on warm water bath (400) containing a small amount of gelatin & taken on
24
oil and grease free glass slides. The slides were air dried and kept in cool place
until staining.
3.9.6. Staining procedure
3.9.6.1. Preparation of Harris hematoxylin solution
Table 7: Preparation of Harris hematoxylin solution
Hematoxylin crystals 5 gm
Alcohol (100%) 50 ml
Ammonium or potassium alum 100 ml
Distilled water 100 ml
Mercuric oxide (red) 2.5 gm
The hematoxylin was dissolved in the alcohol and the alum in the water by the
aid of heat. Two solutions were thoroughly mixed and boiled as rapidly as
possible. After removing from heat, mercuric oxide was added slowly and
reheated to simmer unit it become dark purple. The solution was than removed
from heat immediately and plunged the vessel into basin of cold water until
become cool. The solution was kept in the dark. Immediately before use the
solution was filtered and 2-4 ml of glacial acetic acid was added per 100ml of
solution to increase the precision of the nuclear stain.
3.9.6.2. Preparation of eosin solution (1% stock alcoholic eosin)
Table 8: Preparation of eosin solution (1% stock alcoholic eosin)
Eosin Y, water soluble 1gm
Distilled water 20 ml
Dissolved and 95% alcohol 80 ml
Working eosin solution
Eosin stock solution 1part
Alcohol, 80% 3 parts
25
Flow Chart for Histopathology:
Fixation in neutral buffered formalin
Trimming (0.5-1cm thick)
Overnight washing (8-12 hour)
Dehydration in ascending grades of alcohol
(50%, 1 hour; 70%, 1 hour; 80%, 1 hour; 95%, 1 hour; 100%: 2 changes, 1 hour in each)
Clearing in chloroform/ Xylene
(2 changes, 1 hour and 30 minutes in each)
Embedding in paraffin (3 hour)
Blocking with paraffin
Sectioning on a Microtome
Staining with Hematoxylin & Eosin
Examination under light microscope
26
3.9.6.3. Routine hematoxylin & eosin staining procedure
The tissue sections were deparaffinized in 3 changes of Xyline (3 minutes in each).
Rehydrations of the sectioned tissues were done through descending grades of alcohol.
95% alcohol for 2 minutes
80% alcohol for 2 minutes
70% alcohol for 2 minutes
Distilled water for 5 minutes
Staining with Harris Hematoxylin for 15 minutes
Washing in running tap water for 15 minutes
Differentiated in acid alcohol: 2 to 4 dip (1 part HCl in 99 parts 70% alcohol)
The tissue sections were then washed in tap water for 5 minutes.
Blue in ammonia water (2-3 quick dips)
The sections were then stained with eosin for 1 minute.
27
Differentiation & dehydration in 95% alcohol: 3 quick dips.
The stained sections were then cleaned by 3 changes in Xyline, 5 minutes in each. Finally the sections were mounted with coverslip using DPX.
3.9.6.4. Histopathological studies & photomicrograph
The tissues were examined & photomicrography was taken at the Pathology
Laboratory, BAU, Mymensingh.
3.10. Statistical analysis
Group mean values of weight gain and oocysts output of the chicks were compared by
a students 't' test with N, + N2-2 degrees of freedom (Bailey, 1981).
CHAPTER IV
RESULTS
28
CHAPTER IV
RESULTS
4.1. Mean body weight record
The effect of oral administration of Toltrazuril at different doses were evaluated in
chickens alone or in combination with Vitamin-K, Sulfaclozine, and Oyster mushroom
against chicken coccidiosis (Table 9). The mean initial weight of chicks for all groups
were almost similar, which was 37 g and above as recorded on day 1. The pre-infection
body weight values at day 10 recorded for all groups were almost similar which was
more than 217 g. Significant (P<0.01) increase of body weight was recorded on day 21
following artificial infection in which the highest increase (780.21±1.29 g) was seen in
Group A chicken (healthy control). After treatment (on day 24), there were significant
(P<0.01) increase in body weight of all groups used for therapeutic trial with the
maximum mean weight gain in Group F (850.75±2.40 g) which was the nearest value
to Group A (healthy control) and in Group I (used for prophylactic trial). The chickens
of Group I gained highest (P<0.01) increase in mean body weight (942.60±1.44 g) on
day 24 of the experiment.
4.2. OPG Counts
In Group A, OPG counts remained "0" throughout the experimental period (healthy
control). The initial mean OPG of chickens for other therapeutic trial groups were
almost similar, which was 49 thousands (P<0.01) and above recorded on day 21.
After treatment there were sharp decline of OPG counts in all groups. But the decline
was very significant (P<0.05) in Group F which was 0.09±0.03 thousand. In Group I
(used in prophylactic trial), the pre-treatment value on day 10 and post-treatment on
day 24 were 0 and 0.03±0.01 thousand (P<0.01) respectively (Table 10).
4.3. Mortality and recovery rate record
The clinical signs of caecal coccidiosis appeared on day 20 and became severe on day
21. A total of 3 chicks died during the experimental period from Group B (untreated
affected group) and found 60% mortality. Only one bird died in Group C treated with
Toltrazuril 3.5 mg/kg. In Group I there was no visible morbidity and mortality.
29
After treatment, there was most significant recovery from coccidiosis in Group F with
100% recovery rate. Among Groups B and C, the chicks did not recover as the Group
B was untreated and Group C was treated with low dose of Toltrazuril. The chicks of
Group D, E, G and H had 20, 60, 60 and 80% recovery rate respectively.
4.4. Post-mortem findings after treatment
Group A
Appeared healthy all over the study, lesion was not observed at any part of
gastrointestinal tract (Fig.5).
Group B
The caeca were distended and filled with blood tinged contents, wall showed patchy
haemorrage (Fig. 6 and 7).
Group C, D, E, G and H
There were little haemorrage found on caecal wall with no blood tinged contents
(Fig.8). All other organs were apparently uninfected and normal.
Group F
No significant change was observed in caeca and all other part of intestine (Fig.9).
Group I
In Group I the gastrointestinal tract appeared healthy. The caeca did not reveal any
change.
30
Experimental infection was given at 10 and 15 days of age and therapeutic measure were taken on day 22 and 23 of this study. Prophylactic trial was
set on day 10 of the experiment.
Table 9: Therapeutic and prophylactic efficacy trial of Toltrazuril in broiler chicken in term of body weight gain
Groups Body weight (g)
On day 1 On day 10 (Pre-infection)
On day 21 (Post-infection)
On day 24 (Post-treatment)
Group A (Control) 37.94±0.15 217.24±0.97 780.21±1.29 982.79±3.349 Group B (infected untreated) 37.87±0.38 NS 217.44±1.69 NS 613.59±1.91** 643.97±1.649** Group C (3.5 mg Toltrazuril/ kg body weight) 37.72±0.56 NS 219.74±1.65 NS 616.34±1.93** 749.57±1.422** Group D (7 mg Toltrazuril/ kg body weight) 37.91±0.47 NS 221.24±1.38* 615.00±2.73** 777.03±1.906** Group E (12 mg Toltrazuril/ kg body weight) 38.26± 0.68 NS 220.73±1.50* 618.47±1.56** 765.83±2.10** Group F (7 mg Toltrazuril and 2.5 mg Vitamin K/ kg body weight) 37.85±0.62 NS 222.81±1.36* 615.91±1.96** 850.75±2.40**
Group G (7 mg Toltrazuril and 150 mg Sulfaclozine/ kg body weight) 38.00±0.52 NS 219.88±0.90* 614.60±3.20** 797.22±1.20**
Group H (7 mg Toltrazuril and 100 mg Oyster mushroom/ kg body weight) 37.75±0.57 NS 225.14±1.61** 627.65±1.48** 809.90±1.91**
Group I (used in prophylactic trial) 37.95±0.61 NS 225.72±1.55** 629.53±0.62** 942.60±1.44**
The above values represent the mean ± standard error (SE) of the body weight of 5 chickens *=Significant at 5 percent level (P<0.05) **=Significant at 1 percent level (P<0.01)
31
Table 10: Therapeutic and prophylactic efficacy trial of Toltrazuril in broiler chicken in term of OPG count
Groups OPG count (thousand) Pre-treatment values
(on day 21) Post-treatment values
(on day 24) Group A (Control) 0.00±0.00 0.00±0.00 Group B (infected untreated) 50.55±2.368** 64.44±1.38** Group C (3.5 mg Toltrazuril/ kg body weight) 52.71±1.796** 14.43±1.36** Group D (7 mg Toltrazuril/ kg body weight) 51.65±2.112** 1.58±0.38** Group E (12 mg Toltrazuril/ kg body weight) 49.46±1.33** 1.37±0.27** Group F (7 mg Toltrazuril and 2.5 mg Vitamin K/ kg body weight) 50.44±1.62** 0.09±0.03*
Group G (7 mg Toltrazuril and 150 mg Sulfaclozine/ kg body weight) 46.60±1.86** 1.07±0.04**
Group H (7 mg Toltrazuril and 100 mg Oyster mushroom/ kg body weight) 49.05±0.97** 1.21±0.10**
Group I (used in prophylactic trial) 0.00±0.00NS 0.03±0.01** The above values represent the mean ± standard error (SE) of the body weight of 5 chickens *=Significant at 5 percent level (P<0.05) **=Significant at 1 percent level (P<0.01)
28
Fig. 5: Post mortem examination of the chickens of Group A did not reveal any major
lesion.
Fig.6: Distended caeca filled with blood tinged contents as seen in Group B.
29
Fig.7: Profuse haemorrhage was seen after opening up the caecal tube of Group B
chicken that died of induced coccidiosis.
Fig.8: Haemorrhagic lesion was seen in the mucosa after washing up the caeca of
Group D chicken.
30
Fig. 9: Caeca after washing of Group F showing no lesion on day 24.
31
4.5. Histopathology
At the end of the experiment caeca from the birds of the different groups were
collected for the histopathological study.
Group A
In Group A (control healthy) the caeca showed almost normal structure. Microscopic
lesion was not seen related with caecal coccidiosis (Fig.10).
Group B
The microscopic lesions of the chicks sacrificed on day 24 of this study consisted of
slight thickening of the villi due to infiltration of mononuclear cells and eosinophils in
lamina propria (Fig.11). The glands of the crypt region contain mucous exudates in the
lumen. Merozoites/schizonts were found in contact with villi and crypt epithelium.
Inflammation preceded further additional haemorrhage in the lamina propria and
submucosa (Fig.11). Desquamation of crypt epithelia was found (Fig.11).
Group C
There were erosion and desquamation of crypt epithelia, infiltration of reactive cells in
the lamina propria (Fig.12). Merozoites/schizonts were also found in the crypt of villi
(Fig.12).
Group D
The caeca collected from the chicken of Group D showed arrested coccidial
development with necrosis, haemorrhages, erosion, and desquamation of epithelial
cells (Fig.13).
Group E
There were haemorrages in caecal mucosa and submucosa, infiltration of inflammatory
cells (Fig.14) indicating diffuse acute caecitis.
Group F
Microscopic lesions of caeca of the chicken of Group F showed diffuse infiltration of
reactive cells specially eosinophils and mononuclear cells in the mucosa and
submucosa but haemorrhage was not seen (Fig.15). There was also degeneration of
caecal gland.
Group G
Slight desquamation of caecal epithelia, necrosis, and degeneration of mucosa and
caecal gland were found (Fig.16).
32
Group H
Desquamation of epithelial lining, infiltration of reactive cells in mucosa and
submucosa were found (Fig.17).
Group I
In this group the caeca showed almost normal structure as Group A in which the
histopathology of caeca did not reveal any significant lesion related to coccidiosis
(Fig.18).
33
Fig.10: Caecal section of the chicken of Group A (healthy control) did not reveal any
significant microscopic lesion (H & E, 100X).
Fig.11: Caecal section of the chicken of Group B (infected untreated) showing
desquamation of epithelia, merozoites/schizonts in mucosa, and infiltration of reactive
cells (H & E, 100X).
34
Fig.12: Caecal section of chicken of Group C treated with 3.5 mg Toltrazuril /kg body
weight showing erosion and desquamation of crypt epithelia, infiltration of reactive
cells in the lamina propria with merozoites/schizonts in the crypt of villi (H & E,
400X).
Fig.13: Caecal section of chicken of Group D treated with 7 mg Toltrazuril /kg body
weight showing arrested coccidial development with necrosis, haemorrhages, erosion,
and desquamation of epithelial cells (H & E, 100X).
35
Fig.14: Caecal section of chicken of Group E treated with 12 mg Toltrazuril /kg body
weight showing haemorrages in caecal mucosa and submucosa, infiltration of
inflammatory cells (H & E, 100X).
Fig.15: Caecal section of chicken of Group F treated with7 mg Toltrazuril and 2.5 mg
Vitamin K/kg body weight showing diffuse infiltration of reactive cells specially
eosinophils and mononuclear cells in the mucosa and submucosa, degeneration of
caecal gland but no significant haemorrhage present (H & E, 100X).
36
Fig.16: Caecal section of chicken of Group G treated with7 mg Toltrazuril and 150
mg Sulfaclozine/kg body weight showing slight desquamation of caecal epithelia,
necrosis, and degeneration of mucosa and caecal gland (H & E, 100X).
Fig.17: Caecal section of chicken of Group H treated with7 mg Toltrazuril and 100
mg Oyster mushroom/kg body weight showing desquamation of epithelial lining,
infiltration of reactive cells in mucosa and submucosa (H & E, 100X).
37
Fig.18: Caecal section of chicken of Group I (used as prophylactic trial) treated
with3.5 mg Toltrazuril/kg body weight showing no significant lesion related to
coccidiosis (H & E, 100X).
38
CHAPTER V
DISCUSSION
To have maximum body weight gain in a shortest possible time by spending minimum
feed is the key to success in poultry industry. One of the major constraints having
direct negative impact on body weight gain is coccidiosis in chicken. Karim and Trees
(1990) identified five species of Eimeria (E. tenella, E. necatrix, E. acervullina, E.
maxima and E. brunette) in chicken in Bangladesh. The occurrence of the relatively
less pathogenic species E. precox and E. mitis in chicken in Bangladesh was identified
by Karim and Begum (1994). Thus all the 7 pathogenic species of chicken Eimeria are
present in Bangladesh.
About 104 oocysts of E. tenella once directly introduced into the crop of chicks
normally produce a moderate to severe infection with a very low rate of mortality
(Karim, 1988). Faecal oocyst count and body weight gain were used as the criteria for
the detection of infection and severity of infection. However, faecal oocysts count may
not always correspond with the weight depression, since often a large number of
oocysts may be produced without apparent effect on weight gain (Karim, 1988). Since
the uninfected control chicks had a steady and uninterrupted gain in weight, and did
not pass any oocysts, it can be presumed that the effect on body weight gain was due to
experimental infection. In addition, since the premises, waterer, feeder, brooder etc.
were disinfected with ammonia that kills Eimeria oocysts (Xie et. al. 1983), the
possibility of any additional infection can be excluded.
39
From the body weight records, the maximum mean weight gained was seen in chicks
of non infected healthy chicks (Group A) and was 982.79 g and the minimum mean
weight was achieved (643.97±1.649 g) by the infected untreated chickens of Group B.
The mean weight of other groups remained under this limit. Moreover, it was also
noticed that multiplication of the oocysts administered to test groups was also higher
than usual. The members of Group I (used as prophylactic trial) weighed 942.60 g and
the group was placed in 2nd position. Among the groups of therapeutic trial the
chickens of Group F gained the maximum mean weight which was 850.75 g. The
findings of the present study were correlated with the observation of Lakkundi et al.
(2002), who evaluated the effect of Toltrazuril and amprolium on body weight and
feed efficacy in broiler chickens experimentally infected with caecal coccidiosis. They
found Toltrazuril treated birds had better body weight gain and feed efficiency than
uninfected birds.
The picture of OPG counts in Group I showed that the birds became immune to some
extent after administration of low dose of Toltrazuril (3.5 mg/kg) for 15 days as
prophylactic trail showed range of OPG counts between 0 and 0.03 thousand. This
depicts prophylactic efficacy of the Toltrazuril. However, due to high challenge
infection, the immunity remained fluctuating. The therapeutic trial in different groups
from day 22 to 23 showed mark reduction of mean OPG counts. The maximum
reduction was found in Group F (treated with Toltrazuril 7 mg/kg and Vitamin K 2.5
mg/kg) which was only 0.09 thousand. Similarly, the Group H (treated with Toltrazuril
7 mg/kg and Sulfaclozine 150 mg/kg) had the second position of mean OPG counts
having mean quantity of 1.21 thousand. The present findings substantiated with the
findings of Grief (2000), who claimed that during evaluation studies, Toltrazuril acted
against all intracellular schizonts and being correlated with a higher reduction in
oocyst excretion, lesion scoring and increased weight gains.
From the records of mortality and recovery rate from coccidiosis, it showed that
among the groups of the therapeutic and prophylactic trial the Group F gave the
maximum recovery and lowest mortality rate which were 100 and 0% respectively.
Among the other groups, the Group E, G and H had 60, 80 and 60% recovery rate.
40
Only one bird died in Group C treated with Toltrazuril at a dose rate of 3.5 mg/kg body
weight. This study could be compared with Dhillon et al. (2004), who tested the
efficacy of Toltrazuril against 6 different levels of Eimeria tenella infection in 2-week-
old chicks and found that the treatment resulted to lower mortality and reduced oocyst
production. Moreover, therapy resulted to complete elimination of the clinical signs at
lower levels of infection.
In histopathological study, it was found that the Group A revealed very normal
structure of caecal section as it was used as control group. The group of prophylactic
trial (Group I) also showed normal structure as Group A indicating the prophylactic
efficacy of Toltrazuril. The histopathology of the samples of Group F showed diffuse
infiltration of reactive cells specially eosinophils and mononuclear cells in the mucosa
and submucosa but no significant haemorrhage indicated the anti-haemorrhagic impact
of Vitamin K. In other treated groups (Group C, D, E, G, and H), it was observed that
there were some abnormalities of caeca like infiltration of inflammatory cells in the
mucosa and submucosa, desquamation of crypt epithelia, presence of
merozoites/schizonts in the caecal crypt and villi with only exception in Group E in
which haemorrhage was present prominently in mucosa and submucosa but other
lesions were so prominent. In caecum of Group B, there were slight thickening of the
villi due to infiltration of mononuclear cells and eosinophils in lamina propria, mucous
exudates in the lumen of the glands of the crypt region, merozoites/schizonts in contact
with villi and crypt epithelium. There were also inflammatory infiltrations in additional
to haemorrhages in the lamina propria and submucosa and desquamation of crypt
epithelia indicating the microscopic lesions due to caecal coccidiosis. This study could
be compared with Lakkundi et al. (2002) who evaluated histopathological anticoccidial
activity of Toltrazuril and amprolium in experimentally induced caecal coccidiosis in
broiler chicken (n=280). It was reported that Toltrazuril prevented the establishment of
caecal coccidiosis by degeneration and disintegration of the first generation of
schizonts. It may be concluded that Toltrazuril had a coccidiocidal effect (Lakkundi et
al. 2002).
41
Toltrazuril alone as prophylactic use or use of Toltrazuril in combination with Vit-K or
Sulfaclozine or mushroom can protect chicken from caecal coccidiosis. However, it
needs to study while chickens treated with Toltrazuril, what concentration of
Toltrazuril remain active in the flesh and what public health risk is leying with the
residual value of Toltrazuril in meat and body tissue.
CHAPTER V
DISCUSSION
42
CHAPTER V
DISCUSSION
To have maximum body weight gain in a shortest possible time by spending minimum
feed is the key to success in poultry industry. One of the major constraints having direct
negative impact on body weight gain is coccidiosis in chicken. Karim and Trees (1990)
identified five species of Eimeria (E. tenella, E. necatrix, E. acervullina, E. maxima and
E. brunette) in chicken in Bangladesh. The occurrence of the relatively less pathogenic
species E. precox and E. mitis in chicken in Bangladesh was identified by Karim and
Begum (1994). Thus all the 7 pathogenic species of chicken Eimeria are present in
Bangladesh.
About 104 oocysts of E. tenella once directly introduced into the crop of chicks normally
produce a moderate to severe infection with a very low rate of mortality (Karim, 1988).
Faecal oocyst count and body weight gain were used as the criteria for the detection of
infection and severity of infection. However, faecal oocysts count may not always
correspond with the weight depression, since often a large number of oocysts may be
produced without apparent effect on weight gain (Karim, 1988). Since the uninfected
control chicks had a steady and uninterrupted gain in weight, and did not pass any
oocysts, it can be presumed that the effect on body weight gain was due to experimental
infection. In addition, since the premises, waterer, feeder, brooder etc. were disinfected
with ammonia that kills Eimeria oocysts (Xie et. al. 1983), the possibility of any
additional infection can be excluded.
43
From the body weight records, the maximum mean weight gained was seen in chicks of
non infected healthy chicks (Group A) and was 982.79 g and the minimum mean weight
was achieved (643.97±1.649 g) by the infected untreated chickens of Group B. The mean
weight of other groups remained under this limit. Moreover, it was also noticed that
multiplication of the oocysts administered to test groups was also higher than usual. The
members of Group I (used as prophylactic trial) weighed 942.60 g and the group was
placed in 2nd position. Among the groups of therapeutic trial the chickens of Group F
gained the maximum mean weight which was 850.75 g. The findings of the present study
were correlated with the observation of Lakkundi et al. (2002), who evaluated the effect
of Toltrazuril and amprolium on body weight and feed efficacy in broiler chickens
experimentally infected with caecal coccidiosis. They found Toltrazuril treated birds had
better body weight gain and feed efficiency than uninfected birds.
The picture of OPG counts in Group I showed that the birds became immune to some
extent after administration of low dose of Toltrazuril (3.5 mg/kg) for 15 days as
prophylactic trail showed range of OPG counts between 0 and 0.03 thousand. This depicts
prophylactic efficacy of the Toltrazuril. However, due to high challenge infection, the
immunity remained fluctuating. The therapeutic trial in different groups from day 22 to
23 showed mark reduction of mean OPG counts. The maximum reduction was found in
Group F (treated with Toltrazuril 7 mg/kg and Vitamin K 2.5 mg/kg) which was only
0.09 thousand. Similarly, the Group H (treated with Toltrazuril 7 mg/kg and Sulfaclozine
150 mg/kg) had the second position of mean OPG counts having mean quantity of 1.21
thousand. The present findings substantiated with the findings of Grief (2000), who
claimed that during evaluation studies, Toltrazuril acted against all intracellular schizonts
and being correlated with a higher reduction in oocyst excretion, lesion scoring and
increased weight gains.
44
From the records of mortality and recovery rate from coccidiosis, it showed that among
the groups of the therapeutic and prophylactic trial the Group F gave the maximum
recovery and lowest mortality rate which were 100 and 0% respectively. Among the other
groups, the Group E, G and H had 60, 80 and 60% recovery rate. Only one bird died in
Group C treated with Toltrazuril at a dose rate of 3.5 mg/kg body weight. This study
could be compared with Dhillon et al. (2004), who tested the efficacy of Toltrazuril
against 6 different levels of Eimeria tenella infection in 2-week-old chicks and found that
the treatment resulted to lower mortality and reduced oocyst production. Moreover,
therapy resulted to complete elimination of the clinical signs at lower levels of infection.
In histopathological study, it was found that the Group A revealed very normal structure
of caecal section as it was used as control group. The group of prophylactic trial (Group I)
also showed normal structure as Group A indicating the prophylactic efficacy of
Toltrazuril. The histopathology of the samples of Group F showed diffuse infiltration of
reactive cells specially eosinophils and mononuclear cells in the mucosa and submucosa
but no significant haemorrhage indicated the anti-haemorrhagic impact of Vitamin K. In
other treated groups (Group C, D, E, G, and H), it was observed that there were some
abnormalities of caeca like infiltration of inflammatory cells in the mucosa and
submucosa, desquamation of crypt epithelia, presence of merozoites/schizonts in the
caecal crypt and villi with only exception in Group E in which haemorrhage was present
prominently in mucosa and submucosa but other lesions were so prominent. In caecum of
Group B, there were slight thickening of the villi due to infiltration of mononuclear cells
and eosinophils in lamina propria, mucous exudates in the lumen of the glands of the
crypt region, merozoites/schizonts in contact with villi and crypt epithelium. There were
also inflammatory infiltrations in additional to haemorrhages in the lamina propria and
submucosa and desquamation of crypt epithelia indicating the microscopic lesions due to
caecal coccidiosis. This study could be compared with Lakkundi et al. (2002) who
evaluated histopathological anticoccidial activity of Toltrazuril and amprolium in
experimentally induced caecal coccidiosis in broiler chicken (n=280). It was reported that
Toltrazuril prevented the establishment of caecal coccidiosis by degeneration and
disintegration of the first generation of schizonts. It may be concluded that Toltrazuril had
a coccidiocidal effect (Lakkundi et al. 2002).
45
Toltrazuril alone as prophylactic use or use of Toltrazuril in combination with Vit-K or
Sulfaclozine or mushroom can protect chicken from caecal coccidiosis. However, it needs
to study while chickens treated with Toltrazuril, what concentration of Toltrazuril remain
active in the flesh and what public health risk is leying with the residual value of
Toltrazuril in meat and body tissue.
CHAPTER VI
SUMMERY AND CONCLUSION
CHAPTER VI
SUMMARY AND CONCLUSION
The present research was undertaken to evaluate the therapeutic and prophylactic potentials
of Toltrazuril against chicken coccidiosis in Bangladesh. For therapeutic trial 40 broiler
chicks were divided into eight groups and five in each were used. The infection was induced
by administrating orally about 10,000 sporulated oocyst of Eimeria tenella to chicks of 10
and 15 days of age. Group A was left as noninfected, non-medicated control. Chicks of group
B were infected and non-medicated while chicks of Group C, D, and E were treated with
Toltrazuril at rate of 3.5, 7, and 12 mg/kg body weight on day 22 and 23 of age when the
chicks were severely infected. In chicks of Group F, Toltrazuril was given at rate of 7mg/kg
body with a combination with Vitamin K at the rate of 2.5 mg/kg body weight on same days.
Similarly chicks of Group G were treated with Toltrazuril 7 mg/kg and Sulfaclozine 150
mg/kg body weight and the chicks of group H were treated with Toltrazuril 7 mg/kg and
Oyster Mushroom powder 100 mg/kg body weight. For prophylactic trial the chickens of
Group I were treated with 3.5 mg Toltrazuril/kg body weight before and during exposure to
coccidial infection.
After treatment, the Group F among the groups of therapeutic trial gave the best result on the
basis of body weight, OPG counts, and histopathology. The Group I (used as prophylactic
trial) gave output almost similar to Group A (healthy control).
From the present research it could be concluded that combination of 7 mg Toltrazuril and 2.5
mg Vitamin K/kg body weight would be best solution against caecal coccidiosis than other
doses of Toltrazuril and combination used in the study. This study also suggested that 3.5 mg
Toltrazuril/kg body weight could be of value as prophylactic measure against caecal
coccidiosis.
In this study E. tenella oocysts were used. Further study is needed to determine the efficacy
of Toltrazuril against other species of Eimeria and these researches would provide a clear
idea on the efficacy of Toltrazuril against chicken coccidiosis. Clinical caecal coccidiosis in
chicken can best be treated with Toltrazuril and Vit-K supplement or use of Toltrazuril alone
as prophylactic dose. Use of mushroom with Toltrazuril reduced morbidity and pathologic
lesions following exposure to E. tenella. Analysis is needed to find out the cost benefit
effectiveness of use of the mushroom.
CHAPTER 5
REFERENCES
48
REFERENCES
AL-Taee, S. N., Zenad, K. N. and Khamas, W. A. (1993). Pathology of experimental
infection by E. tenella in broiler chicks treated with monensin or maduramicin.
Pakistan Veterinary Journal 13(1): 34-36.
Amarjit Singh; Grewal, GS and Singh, A. (1994). Patho morphological changes due to
rickets in chicks. Indian Journal of Animal Sciences 64(11): 1132-1137.
Anon (2006). Report of BRAC on Avian Influenza Prevention Programme. November
2005- August 2006, p-1.
Anonymous (1999). The fifth five year plan. Planning commission, Ministry of
planning, Government of the peoples Republic of Bangladesh.
Bailey, N. T. J. (1981). The use of t-test for small samples. Statistical Mothods in
Biology. 2nd Edn. Hodder and Staughton, Great Britain. . Pp. 43-51
Barnes, H. J., Calnek, B. W., Reid, W. M. and Yoder, H. W. (1992). Diseases of
Poultry. 8th Bd. Edited by Hofstad, M. S. The Iowa state University Press,
Iowa.
Bhattacharijee, P. S., Kundu, R. L., Biswas, R. K., I`viazumder, J. U., Hossain, E. and
Miah, A. H. (1996). A retrospective analysis of chicken diseases diagnosed at
the central Disease Investigation Laboratory, Dhaka. Bangladesh Veterinary
Journal 30(3-4): 105-113.
Borchers A. T., Krishnamurthy A, Keen C. L., Meyers F. J., Gershwin M. E. (2008).
The immunobiology of mushrooms. Experimental Biology and Medicine 233:
259-276.
49
Bollengier, L., Williams P. E. V. and Whitehead C. C. (1999). Optimal dietary
concentration of vitamin E far alleviating the effect f heat stress on egg
production in laying hens. British Poultry Science 40: 102-107.
Bottje, W., Enkvetchal.ul, B., Moore, R. and McNew, R. (1995). Effect of cc-
tocopherol on antioxidants, lipid peroxidation and the incidence of pulmonary
hypertension syndrome (ascites) in broilers. Poultry Science 74: 1356-1369.
Claeskens, M., Verdonck, W., Heesen, H., Froyman, R. and Torres, A. (2007). A field
study assessing control of broiler coccidiosis by
Paracox<ovid:sup>TM</ovid:sup> vaccination or by toltrazuril (BaycoxReg.)
stand-alone treatment. Parasitology-Research 101(Supplement.1): S105-S112
Chapman, H. D. (1978) E. tenellu; experimental studies on the development of
resistance to amprolium, clopidol and methyl benzoquate. Parasitology 76:
177-183.
Chapman, H. D. (1982a). The treatment of coccidiosis: Studies on the sensitivity of
recent field isolates of E. acervulina type to anticoccidial drugs given in the
drinking water. Journal of Comparative Pathology 92(2): 213-218.
Chapman, H: D. (1982b). The sensitivity of field isolates of E. acervulina type to
monensin. Veterinary Parasitology 9(3/4): 179-183.
Chapman, H. D. (1983). Field isolates of Eimeria resistant to aprinocid. Veterinary
Parasitology 12(1): 45-50.
Chapman, H. D. (1984). E. tenella: experimental development of resistance to
rnonensin in the chicken. Parasitiology 89(1): 9-16.
Chapman, H. D. (1986). Isolates of E. tenella: studies on resistance to ionophorous
anticoccidial drug. Research in Veterinary Science 41(2): 281-282.
50
Chapman, H. D. (1987). Control of a line of E. tenella, partly resistant to monensin, by
including Toltrazuril discontinbuously in drinking water of chickens. Journal of
Comparative Pathology 97(1):21-27.
Dam, H. (1942). Vitamin K, its Chemistry and Physiology. Advances in Enzymology
and Related Areas of Molecular Biology, Volume 2 (eds Nord F. F. and
Werkman C.)
Das, P. M., Rajab, D. M. M., Noor, M. and Islam, M. R. (2005). Retrospective analysis
on the proportional incidence of poultry diseases in greater Mymensingh
district of Bangladesh. In: proceeding of 4th International Poultry Show and
Seminar. The World Poultry Science Association, Bangladesh Branch, pp. 35-
39.
Danforth, H. D. (1986). Use of hybridoma antibodies combined with genetic
engineering in the study of protozoan parasite: a review In: Research in Avian
Coccidiosis. I. R. 49
Dikovskaya, V. E. (1974). Intraspecies variability in Eimeria tenella in the chicken. (In
Russian) Parazitologiia 8: 548552.
Dhillon, N. K., Jasmer, S., Bal, M. S. and Ashuma, T. (2004). Efficacy of toltrazuril
against different levels of Eimeria tenella infection in chicken. Journal of
Research, Punjab Agricultural University 41(1): 150-155.
Doran, D. J. and Vetterling, J. M. (1969). Influence of storage period on excystation
and development in cell culture of sporozoites of Eimeria meleagrimitis Tyzzer,
1929. Proceedings of the Helminthological Society of Washington 36(1): 33-
35.
Edgar, S. A. (1955). Sporulation of oocysts at specific temperatures and notes on the
prepatent period of several species of avain coccidia. The Journal of
Parasitology 41: 214-216.
51
El-Banna, H. A., EI-Bahy , M. M. , EI- Zorba, H.Y. and EI- Hady, M. (2005).
Anticoccidial efficacy of drinking water soluble diclazuril on experimental and
field coccidiosis in broiler chicken. Journal of Veterinary Medicine 52(6): 287-
291.
Erasmus, J., Scott, M. L. and Levine, P. P. (1960). A relationship between coccidiosis
and vitamin A nutrition in chickens. Poultry Science 39: 565-72.
FAO (2002) Global product of meat, milk and eggs (million of metric tones) in the
developed and developing countries, Statistical Database, 2002.
Ghazala, N., Qazi, A.W., Gill, Z. J., Rizwan, Q. and Sajid, M. A. (2003). Evaluation of
2.5 percent Toltrazuril against coccidiosis in broiler chicks. Pakistan Journal of
Veterinary Research 1(2): 8-11
Giasuddin, M., Alam, J., Islam, M. R. and Rahman, M. M. (2003). Epidemiological
investigation of infectious Bursal disease in Bangladesh. Presented in the 3rd
International poultry show and seminar, Dhaka, Bangladesh. February 28-
March 2, 2003. pp 34-37.
Giasuddin, IVI., Sil, B. K., Alam, J., Koike, I., Islam, M. R.and Rahman, M. M.
(2002). Prevalence of poultry diseases in Bangladesh. Journal of Biological
Science 2(4): 212-213.
Ghanem, M. M., Radwaan, M. E., Moustafa, A-M. M. and Ebeid, M. H. (2008) :
Comparative therapeutic effect of toltrazuril, sulphadimidine and amprolium on
Eimeria bovis and Eimeria zuernii given at different times following infection
in buffalo calves (Bubalus bubalis). Preventive Veterinary Medicine 84: 161-
170.
Gill, B. S. and Bajwa, R: S. (1979). Drug resistance in field isolates of chicken
coccidia from Punjab State. Indian Journal of Parasitology 3(2): 131-134.
52
Gjerde, B. and Helle, O. (1991). Chemoprophylaxis of coccidiosis in lambs with a
single dose of toltrazuril. Veterinary Parasitology 38(2-3):97-107.
Goodrich, H. P. (1944). Coccidian oocysts. Parasitology 36: 72-79.
Grief, G. Stephan, B. and Haberkorn, A. (1996). Intraspecific polymorphisms of
Eimeria species due to resistance against anticoccidial drugs. Parasitology
Research 82(8): 706-714.
Grief, G. (2000). Immunity to coccidiosis after treatment with toltrazuril. Parasitology
Research 86 (10): 787-790.
Grilli, G., Giussani, N., Ceruti, R., Gavazzi, L., Pisoni, AM; Ferrazzi, V; Gallazzi, D
(2003). Use of toltrazuril treatment for the control of broiler coccidiosis. Large
Animals Review 9(6): 123-124.
Guo Y. M., Zhang G. M., Yuan J. M. and Nie W. (2003). Animal Feed Science and
Technology 107(1-4): 143-150.
Haq, A. L., Bailey, C. A. & Chinnah, A. (1996). Effect of (3carotene, canthaxanthiri
supplemented in the broiler breeder diets. Poultry Science. 75: 1092-1097.
Harder, A. and Haberkorn, A. (1989). Possible mode of action of toltrazuril: studies on
two Eimeria species and mammalian and Ascaris suum enzymes. Parasitology
Research 76 (1): 8-12.
Harms R. H., Waldroup, P.W. and Cox, D. D. (1960). Comparison of Various Sources
and Levels of Vitamin K Activity Using Chicks with Cecal Coccidiosis.
Experimental Biology and Medicine 105:230-232
Hodges, J. N; Bal, S. J; Ryan, K. C. and Warren, E. W. (1969). The incidence of drug
resistant strains of Eimeria in chicken in Great Britain. British Veterinary
Journal 125: 31-35.
53
Hoffmann-La Roche. (1972). Vitamin E, No. 1206. F. Hoffmann-La Roche & Co.
Ltd., Basel, Switzerland.
Hou ShuiSheng; Tong JianMing; He ShengChai; Huang JunCun; Hou SS; Tong JM;
He SC and Huang JC. (1999). Influence of vitamins A, D and E on the growth
and tissue vitamin content of broilers. Acta Nutrimenta Sinica 21(4): 428433.
Huan J; cheeke PR; Lowry RR; Nakaue HS; Snyder SP and Whanger PD. (1992).
Dietary pyrrolizidine (Senecio) alkaloids and tissue distribution of copper and
vitamin A in broiler chickens. Toxicology Letters 62: 2-3, 139-153.
Huque, Q. M. E. (2001). Poultry industry in Bangladesh and strategies for its
improvement. In: proceeding of 2th International Poultry Show and Seminar.
The World Poultry Science Association, Bangladesh Branch, pp. 34-39.
Islam, M. K.; Mondal, M. M. H, and. Quadir, A. N. M. A: (1996). Prevalence and
distribution of coccidiosis in dead chicken. The Bangladesh Veterinarian 13(1-
2): 32-33.
Jedinak, A, Dudhgaonkar, S, Wu, Q, Simon, J and Sliva, D. (2011). Anti-
inflammatory activity of edible oyster mushroom is mediated through the
inhibition of NF-κB and AP-1 signaling. Nutrition Journal 10:52.
Jedinak, A, Sliva, D. (2008) Pleurotus ostreatus inhibits prolifieration of human breast
and colon cancer cells through p53-dependent as well as p53-independent
pathway. International Journal of Oncology 33: 1307-1313.
Jeffers, T. K. (1974). E. acervulina and E. maxima: Incidence and anticoccidial drug
resistance of isolates in major broiler production areas. Avian Diseases 18: 331-
342.
54
Jeffers, T. K. (1978). Genetics of Coccidia and the host response. In: Long P. L,
Boorman, K. N. and Freeman, B. M. (eds) Avian Coccidiosis, 13th Poultry
Science Symposium. British Poultry Science Ltd; Edinburgh, pp. 50-125.
Jeffers, T. K. and Bentley, E. J. (1980). I. Monensin sensitivity of recent field isolates
of Turkey Coccidia. lI. experimental development of monensin resistance in E.
meleagrimitis. Poultry Science 59(8): 1722-1735.
Jin-GuangMing; Cheng-CongSheng; Gu-YouFang; ChenHuiLiang; Wu-QiHong; Jin-
GM; Cheng-CS; Gu-YF; ChenHL; Wu-QH. (2003). Resistance of two Eimeria
tenella isolates to five anticoccidiosis drugs. Chinese Journal of Veterinary
Science 23: 1, 40-42;
Joyner, L. P. (1970). Coccidiosis: Problem arising from the development of
anticoccidial drug resistance. Experimental Parasitology 28: 122-128.
Jungmann, R. and Mielke, D. (1989). Immunoprophylaxis of coccidiosis in poultry
with an irradiated Eimeria tenella vaccine. Monatshefte fur Veterinarmedizin,
44(13): 464466.
Kannan; Y; Nishio, S. and Kato, S. (1995). Effects of dietary calcium levels on the
histomorphology of tibial diaphysis in vitamin D deficient chicks. Japanese
Poultry Science 32(2): 107-116.
Karim, M. J. (1988). lntra specific variations in avian coccidia of British and
Bangladeshi origin. Ph.D. Thesis submitted to the University of Liverpool,
England, August 1988. p. 34.
Karim, M. J. and Trees, A. J. (1990). Isolation of five species of Eimeria from
chickens in Bangladesh. Tropical Aninaal Health and Production 22(3): 153-
159.
55
Karim, M. J. and Begum, N. (1994). Morphological and biological characterization of
chicken Eimeria with special reference to species identification. Veterinary
Review 9(1): 7-9.
Karim, M. J., Begum, N. and Khan, M. S. R. (1994). Prevalence of coccidiosis in
broilers on two farms in Bangladesh. Pakistan Journal of Scientific and
Industrial Research 37(8): 339-341.
Karim, M. J. (2001). Coccidiosis in chicken in Bangladesh= Present status and
strategies for control. Proceedings of the 2"d Int. Poultry show and seminar,
February 16-17, 2001. p-109-116.
Krylov, V. F. and Zaionts, V. I. (1981). The prevalence of drug resistant cultures of
coccidia. Veternariya Moscow 3: 4546.
Kutubuddin, M. A. (1973). Pathological investigation on the cases of mortality of
poultry birds in the BAU poultry farm. M. Sc. Thesis. Department of
Pathology, Bangladesh Agricultural University, Mymensingh.
Lakkundi-JN; Jagannath-MS; D'-Souza,-PE. (2002). Effect of toltrazuril and
amprolium on body weight and feed efficiency of broiler chicken
experimentally infected with Eimeria tenella. Indian Journal of
Animal,Sciences 72(10): 835-837.
Lakkundi-JN; Jagannath-MS; D'-Souza,-PE. (2002). Histopathological evaluation of
anticoccidial activity of toltrazuril and amprolium in experimental Eimeria
tenella infection in chicken. Indian Journal of Animal,Sciences. 2002. 72(5):
372-374.
.
Long, P. L. (1973). Pathology and pathogenicity of coccidial infections. In: The
coccidia: Eimeria, Isospora, Toxoplasma and Related Genera. (Eds. Hammond;
D. M.; Long, P. L.) pp. 253-294. Baltimore: University Park Press.
56
Long, P. L. (ed) (1982). The Biology of the coccidia. Univ. Park Press, Baltimore,
USA.
Long, P. L. and Jeffers T. K. (1986). Anticoccidail medication programme vary for
broilers, cage layers and breeders. Feed Stuffs 58(25): 34-37.
Manzi, P., Gambelli, L., Marconi, S., Vivanti, V. and Pizzoferrato, L. (1999). Nutrients
in edible mushrooms: an inter-species comparative study. Food Chemistry
65(4): 477-482.
Mathis, G. F. and McDougald, L. R. (1982). Drug responsiveness of field isolates of
chicken coccidia. Poultry Science 61(1): 38-45.
Mathis, G. F., Froyman, R. and Kennedy, T (2004). Coccidiosis control by
administering toltrazuril in the drinking water for a 2-day period. Veterinary-
Parasitology 121(1/2): 1-9
Maynard, L. A., Loosli, J. K., Hintz, H. F. and Warner, R. G. (1979). Animal Nutrition,
7th 'Ed. McGraw-Hill, New York. p. 283.
Mehlhorn, H., Ortmann-Falkenstein, G. and Haberkorn, A. (1984). The effects of sym.
Triazinones on developmental stages of Eimeria tenella, E. maxima and E.
acervulina: a light and electron microscopical study. Parasitology Research 70
(2):173-182.
Mehlhorn, H., Schmahl, G. and Haberkorn, A. (1988). Toltrazuril effective against a
broad spectrum of protozoan parasites. Parasitology Research 75 (1): 64-66.
McDougald, L. R. (1981). Anticoccidial drug resistance in the South-eastern United
States: Polyether, Ionophorous drugs. Avian Diseases 25: 600-609.
57
Mondal, M. M. H., Qadir, A. N. M. A. (1978). A preliminary investigation on the
incidence of coccidial investigation in fowl, goat and cattle. Bangladesh
Veterinary Journal 12:7-11.
Munoz, L. and Rodriguez, V. (1988). Comparative activity of ionophores against E.
acervulina-mivat+E. tenella isolated in commercial farms in Europe. In Vet.
Pharmacology, Toxicology and Therapy in food producing animals. 4th
congress of European, Budapest, Hungar, August 28-September 2, 1988.
Volume l,/Abstract.
Murphy, R. R., Hunter, J. E. and Knandel, H. C. (1938). The effect of rations
containing gradient amounts of cod liver oil on the subsequent performance of
laying pullets following a natural infection of coccidiosis. Poultry Science 17:
377-80.
Nayak, D. C. and Rai, P. (1985). Haemogram of chickens experimentally infected with
Eimeria species. Indian Journal of Veterinary Medicine, 5(1): 42-43.
National Research Council (1994). Nutrient Requirements of Domestic Animals.
National Academy of Sciences. National Research Council, Washington, D. C.
Nutrient Requirements of Poultry, 9th Edition.
Oikawa, H. Kawaguchi, H., Nakamoto, K. and Tsunoda, K. (1975). Survey on drug
resistance of chicken coccidia collected from Japanese broilers farms in 1973.
Japanese Journal of Veterinary Science 37(4): 357-362.
Raha, S. K. (2007). Broiler industry in Bangladesh: some issues. In: proceeding of 5th
International Poultry Show and Seminar. The World Poultry Science
Association, Bangladesh Branch, pp. 1-9.
Rao-NS. (I988). Vitamin A deficiency in poultry. Poultry Adviser 21(5): 47-48.
58
Ramadan, A., Abo El-Sooud, K. and El-Bahy, M. M. (1997). Anticoccidial efficacy of
toltrazuril and halofuginone against Eimeria tenella infections in broiler
chickens in Egypt. Research in Veterinary Science 62 (2): 175- 178.
Reather, W. and Paeffgen, D. (1989). Drug sensitivity of coccidia recently selected
from broiler farms in Europe. In: Coccidia and Intestinal Coccidiomorphs.
Proceedings of the 5{h International Coccidiosis Conference. Tours (France),
17-20 October 1989.
Roberson KD. (1992). Effects of calcium, vitamin D metabolites and insulin like
growth factor I on the epiphyseal growth plate of broiler chickens. Proceedings
of the Georgia Nutrition Conference for the Feed Industry. ; Atlanta Airport
Hilton, Atlanta, Georgia, USA, November 17-19.
Rommel, M. (1987). Prophylaxis and therapy of avian coccidiosis Berliner und
Munchener Tierarztliche Wochenschrift 100(8): 270-272. Proto-zoological
Abstracts (1988). Abstract No. 883.
Ryley, J. F. (1973). Techniques (oocyst isolation). In: the coccidia. (eds. Hammond, D.
M.; Long, P. L.) University Park Press, Baltimore.
Ryley, J. F.,Hardman, L. (1978). The use of vitamin K deficient diets in the screening
and evaluation of anticoccidial drugs. Parasitology 76:11-20
Ryley, J. F. (1980). Drug resistance in coccidia. Advance in Veterinary Science and
Comparative Medicine 24: 99-100.
Safonova, I., Amri, E. and Ailhaud, G. (1994). Molecular and Cellular Endocrinology
104, 201.
Saleque, M. A. (2003). Pattern of poultry diseases in Breeding farm and poultry units
in Bangladesh. Presented in the 3Ta World Poultry show and seminar, Dhaka,
Bangladesh. February 28- March 2, 2003. pp 7-10.
59
Saleque, M. A. (2006). Poultry Industry in Bangladesh: current status and its future.
Poultry Business Dictionary. Poultry Khamar Bichitra, p 25.
Salisch (1987). Efficacy of the anticoccidial maduramicin ammonium under
experimental ;ind field condition. Archiv fur Geflugelkunde 51(5): 190-196.
Protozoologial Abstracts (1988). Abstract No. 1455.
Salisch, H. and Friederichs, M. (199I). Efficacy of salinomycin and maduramicin
against E. bruetti in chicken Archiv fur Geflugelkunde 55(2): 61-63.
Protozoological Abstracts (1992). Abstract No. 2857.
Salisch, H. and Shakshouk, A. G. R. (1989). Efficacy of diclazuril and four inophorous
anticoccidials against E. tenella and E. acervulina. Archiv fur Geflugelkunde
53(3): 111-115. Protozoological Abstracts (1989). Abstract No. 3833.
Sanei, M. M., Rahbari, S., Mansouri, B. and Kiaei S.M.M. (2009). The effect of
sulfaclozine 30% (Esb3) on experimental coccidiosis in broiler cockerels.
Scientific-Research Iranian Veterinary Journal 5(2 (23)):62-69
Sanda, A. (1986). Vaccination of chicks against cocidiosis on large farms.
Veterinarstvi 36(8): 371-372.
Sarker, A. J. (1976). The prevalence of avian disease in Bangladesh Agricultural
University Poultry Farm. Bangladesh Veterinary Journal 10(1/4): 61-66.
Sato, Y; Schneebeli, M and Sato, G. (1992). An occurrence of vitamin A deficiency in
chickens in Zambia. Journal of Veterinary Medical Science 54(3): 601-603.
Sentape, I. and Eraslan, G. (2010). Pharmacokinetic of sulfaclozine in broiler chickens.
Food and Chemical Toxicology 48(1): 448-451.
Scott, M. L., Nesheim, M. C. and Young, R. J. (1982). Nutrition of the Chicken, Scott,
Ithaca, New York.
60
Seeripto, (1984). Eimeria tenella infection in chickens. Penyakit Hewan. 16(27): 169-
172.
Sen, M. M., Karunanidhi, P. S., Grewal, G. S. and Mishra, S. K. (1981). An
investigation into coccidiosis in chicken. Poultry Adviser 14(4): 65-67.
Sheng-chao, F., Hui-juan, Y., Jin-wei, C. and De-nian, M. (2005). Efficacy Evaluation
of Combinations amongSelected Anticoccidials against Eimeria tenella in
Chicken. Acta Agriculturae Shanghai 21 (1): 109-113.
Shafey; NicDonald and Pym RAE. (1990). Effects of dietary calcium, available
phosphorus and vitamin D on growth rate, food utilisation, plaama and bone
constituents and calcium and phosphorus retention of commercial broiler
strains. British Poultry Science 31(3): 587-602.
Sharif, M. (2002). CuTrent status of chicken coccidiosis in Gazipur and coccidiosis
control in semi-scavenging layer pullets. M. S. Thesis. Submitted to Dept. of
Parasitology, BAU, Mymensingh.
Shirley, M. W: and Millard, B. J. (1986). Studies on the pathogenicity of seven
attenuated lines of Eimeria given as a mixture to chickens. Avian Pathology.
15(4): 629-638.
Shirley, M. W. (2000).The genome of Eimeria spp. with special reference to Eimeria
tenella-a coccidium from the chicken. International Journal Parasitology 30,
485-493.
Siddiki, A. M. A. M. Z., Karim, M. J., Islam, M. K. and Alam, M. Z. (2000).
Sulphonamide resistance in field isolates of chicken coccidia in Bangladesh.
The Bangladesh Veterinarian 17(1) pp. 11-15.
61
Simpkins and Fried B. (1999). Effects of a diet deficient in vitamins A, D and E on
infectivity, growth and development of Echinostoma trivolvis in domestic
chicks. Parasitologv Research 85(10): 873-875.
Sklan, D., Nlelamed, D. & Friedman, A. (1994). The effect of varying levels of dietary
vitamin A on immune response in the chick. Poultry Science 73: 813-847.
Stephen, J. F., Tugwell R. L. (1960). Sources and Levels of Vitamin K in Relation to
Cecal Coccidiosis. Poult. Sci.. 39(5): 1183-1187.
Stephen, B., Rommel, M., Dangschies, A. and Haberkorn, A. (1997). Studies of
resistance to anticoccidials in Eimeria field isolates and pure Eimeria strains.
Veterinary Parasitology 69: 19-29.
Talha, A. F. S. M. (1999). Pathology of poultry diseases occurring in Mymensingh. M.
S. Thesis. Dept. of Pathology. Bangladesh Agricultural University,
Mymensingh, 2002.
Taylor, M. W. and Russela, W. C. (1947). The pro-vitamin A requirement of growing
chickens Poultry Science 26: 13140.
Trees, A. J. (1987). Parasitic conditions in poultry. 1. Protozoological Diseases, In
Practice 9: 86-89.
Tyzzer, E. E. (1929). Coccidiosis in gallinaceous birds. American Journal of Hygiene.
10: 269-383.
Uni, Z., Zaiger, G.,Gal Garber, O., Pines, M., Rozenboim, I. and Reifen, R. (2000).
Vitamin A deficiency interferes with proliferation and maturation of cells in the
chicken small intestine. British Poultry Science 41(4): 410-415.
Varga, I, Laczay, P. and Voros, G. (1988). Anticoccidial efficacy of maduramicin in
the battery and floor per trials. In: Veterinary pharmacology, toxicology and
therapy in food producing animals. 4th congress of European Association for
62
Veterinary Pharmacology and Toxicology, Budapest, Hungary, August 28
September 2, 1988. Volume 1. Abstracts.
Vervelde, L. and Vermeulen, A. N. (1995). Eimeria Tenella:Sporozoites rarely enter
leukocytes in the cecal epithelium of the chicken (Gallus domesticus).
Experimental Parasitology 81(1): 29-38
Villamide, M. J. and Fraga, M. J. (1999). Composition of vitamin supplements in
Spanish poultry diets. British Poultry Science. 40: 644-652.
Waldenstedt L; Elwinger K; Lunden A; Thebo P and Uggla A. (2000). Broiler
performance in response to a low protein or a vitamin supplemented diet during
experimental coccidial infection. Archiv fur Geflugel kunde 64(1), 3439.
Waldroup, P. W., Simpson, C. F. Cox, D. D. and Harms, R. H. (1963). The effects of
feeding various levels of vitamin A on chicks with cecal coccidiosis. Poultry
Science 42: 27475.
Ward NE. (1993). Vitamin supplementation rates for U.S. commercial broilers;
turkeys, and Layers. Journal of Applied Poultry Research 2(3): 286-296.
Warren, E. W., Ball, S. J. and Mackenzie, D. R. (1966). The incidence of Drug-
Resistant strains of Eimeria species in chickens in Great-Britain. British
Veterinary Journal 122: 534-543.
Warren, E. W. (1968). Vitamin requirements of the Coccidia of the chicken.
Parasitology, 58: 137-148
Wasser S. (2002). Medicinal mushrooms as a source of antitumor and
immunomodulating polysaccharides. Applied Microbiology and Biotechnology
Volume 60, Number 3, 258-274.
Watkins; Garnsworthy and Cole,DJA. (1993). Diet and leg weakness in poultry. Recent
Advances in Animal Nutrition 131-142.
63
Waxlen, S. H. (1941). Changes occurring in the blood and tissues of chickens during
coccidiosis and artificial haemorrhage. The Amarican Journal of Physiology
134: 19-26.
Whanger, P. D. (1981). In Selenium in Biology and Medicine; (J. E. Spallholz, J. L.
Martin, and H. E. Ganther, eds.), p. 230. AVI, Westport, Connecticut.
Wiedermann, U., Hanson, L. A., Holmgren, J., Kahu, H. and Dahlgren, U. I. (1993).
Infection and Immunity 61: 3952.
Wojcik S; Adamczyk M and Niedzwiadek T. (2001). The effect of some nutrients on
growth of chicks after hatching. Biuletyri Naukowy Przemyslu Paszowego 40(1-
4): 5-15.
Xie, M. Q., Lu, Y. M. and Wen, L. N. (1983). Coccidiostatic and coccidiocidal test on
coccidial oocysts of chickens. Chinese Journal of Veterinary Medicine 9(10): 2-
5.
Yun, C. H., H.S. Lillehoj and E. P. Lillehoj (2000). Intestinal immune responses to
coccidiosis. Developmental and Comparative Immunology 24(2-3): 303-324.
Zhang, Z. and Zeng, M. (2005) : Researches advance of drug resistance in chickens
coccidian. Chinese Journal of Veterinary Parasitology 13: 29-36.
Zhang Qin, Liu SuQian, Li Ruixue, Zhang, Jin Jiasg, Liu JueRu. (1995). Efficacy of an
aqueous solution of maduramicin against E. tenella. Chinese Journal of
Veterinary Science 25(11): 41-43.