UNIVERSITY OF AGRICULTRE, FAISLABAD

FACULTY OF VETERINARY SCIENCE

DEPARTMENT OF PARASITOLOGY
(SYNOPSIS FOR M.Phil. DEGREE)
Title: Evaluation of In vivo and In vitro anticoccidial activity of Trachyspermum ammi
Essential Oil
Name of the student : M. Rashid Fayyaz
Registration #: 2010-ag-2424

Abstract

Date of admission: 06-09-2016

Probable duration: 4 months

Personnel:
Name of student: M. Rashid Fayyaz
Registration No: 2010-ag-2424

Supervisory Committee:
1. Dr. Rao Zahid Abbas (Chairman)
2. Dr. Zia ud Din Sindhu (Member)
3. Dr. Muhammad Kashif Saleemi (Member)

Need of Project:
In our country the 19 % meat production is subsidized by poultry industry that is the best source
of animal protein(Ahmad et al., 2010). The demand of poultry industry is increasing every day but
due to parasitic diseases like coccidiosis is facingerious problems all over the world (Saima et al.,
2010). In commersialized system of poultry farming the coccidiosis ranges from 5-70% (Du and
Hu, 2004).
Coccidiosis is the protozoa born highly pathogenic disease of poultry caused by Eimeria Genus
(Abbas et al., 2015). Coccidiosis is one of the major threats to poultry industry worldwide and it
is responsible for heavy economic losses that could reach upto $ 3billion per annum(Kadykalo et
al., 2017) The economic losses are more prominent in developing countries like Pakistan and
according to estimate coccidiosis led to $127 million losses to poultry industry in united state
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(Bachaya et al., 2015; Chapman, 2014) These economic losses caused by coccidusis resulted due
to mal-nutrient absorption (Shirley and Lillehoj, 2012) reduced weight gain, bloody diarrhea, poor
FCR and dehydration (Lillehoj and Lillehoj, 2000; McDougald, 1998). Coccidia infection occur
due to shedding of oocyst in the environment and ingestion of sporulatedoocysts, infected litter,
contaminated feed as well as bad watering system make favorable conditions to proliferate
infection in poultry birds in subclinical form. Coccidiosis becomes very much difficult to be
eliminated it after infection has spread in clinical form (Bachaya et al., 2015; Sundar et al., 2017)
Traditionally coccidiosis is being controlled by using anticoccidial drugs as feed additive from
1940 as preventive measures. However problem is that uncritically and frequently use of these
drugs developed resistance in chickens against genus Eimeria. The commercially available
anticoccidials proved to be unproductive to control coccidiosis(Abbas et al., 2011; Abbas et al.,
2008; Blake and Tomley, 2014; McDougald and Reid, 2003).The continuous usage of
anticoccidial drugs lead to the toxic effects upon health of birds as well residual effects of these
drugs in mutton compelled the researchers to find out alternative method to control poultry
coccidiosis(Abu-Akkada and Awad, 2015; Sundar et al., 2017; Yang et al., 2015). Vaccines
provide an alternative for disease protection and they ultimately help in reducing resistance against
Eimeria. Combination of vaccination with botanicals and probiotics provided additional and
enhanced protective effect against coccidiosis in broiler chickens ((Ritzi et al., 2016)
The plant driven products can act as best alternative source to avoid anticoccidials drugs for control
of coccidiosis in poultry industry. There has been an emerging interest by using plant essential oils
to control coccidiosis due to anticoccidials resistance issue in poultry industry as well burning
concern about the residues in meat byproducts. Therefore using Essential oils against coccidiosis
is profitable approach over anticoccidials drugs. The use of essential oils against coccidiosis is
limited due to their antinutritional , cytotoxic effects, under dose response and issues in
commercial availability. However their complexity in structure, approach in clearity in mechanism
of action the essential oils are being used to improve control strategies for coccidiosis. Therefore
before using plant essential oils one should be clear about the secondry metabolic products as well
potential of ant nutritional factors (Akthar et al., 2014). in such a process that they should no
compensate the required anticoccidial effects. The current study planned to determine the potential
of essential oils being used as anticoccidials.

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The specific goal of this study will be
1. To determine the anticoccidial activity of Essential oil of
Trachyspermum ammi of Seed
2. To evalaute the effect of Essential oil of Trachyspermum ammi on
hematological parameters and serum chemistry of infected chicks

Review of literature

Plants produce many phytochemical products from primary as well secondary metabolism out of
many having medical importance like antimicrobial (antiviral, antibacterial, antifungal and ant
parasitic) properties (Singh, 2017).Most reportable and valuable plants possessing antiparasitic
activity belong from the aromatic families including Apiaceae, Lamiaceaeand Asteraceae(Abbas
et al., 2012a; Abbas et al., 2012b; Christaki et al., 2012; Zhai et al., 2007)
From plants metabolic byproducts the Essential oils are the naturally occurring complex impulsive
compounds having durable odor have been used in Arabs in first time by using steam distillation
(Cosentino et al., 2003; Guenther, 1948)Essential oils obtained from various aromatic plants
present in temperate, tropical and subtropical regions having great value in unani medicine in past
decades to now a day.Essential oils occur in various forms like liquid, volatile and sometimes
colouredorganic substances having density lighter as compare to water. Essential oils con be
obtained from stem, leaves, barks, roots, buds and flowers of plants that are being used in medicinal
practice (Angioni et al., 2006; Masotti et al., 2003). Mostly it is supposed that immunomodulatory,
antifungal, insecticidal and antimiocrobial activity(Khater, 2012; Khater and Khater, 2009) of
essential oils are directly associated with their antiparasitic and specially anticoccidial
activity(Khater, 2014). The anticoccidial activity (Liu et al., 2007; Zhai et al., 2007)of single
component of essential oil is more precisely reported than the whole essential oil. In Eimeria
infection (Giannenas et al., 2004; Williams et al., 1997)the phenolic components were studied by
the reserchers concluded that these components directly change the permeability of cytoplasmic
membrane to various cations especially for potassium and hydrogen ions. This change in the
permiabilty lead to the hinderence at cellular level for example loss of cellular components ,
inhibition of ATP synthesis ultimately led to cellular death of the disease causing
organism(Christaki et al., 2004; Giannenas et al., 2004; Giannenas et al., 2003; Ultee et al., 1999).

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The essential oils obtained from the Allium CepEchinacea
purpure,ChenopodiumambrosioidesMentha Species as well Allium sativumhave beed mentioned
valuable results against the Eimeria species(Zhai et al., 2007).The EOS obtained from the
O.vulgare have been reported against E.tenella, (Silva et al., 2009)E. maxima), E. acervulina(Ibrir
et al., 2001)as well as for mixed Eimeria infection (Hume et al., 2006)
The various components of essential oils have been widely reported positive response for
controlling coccidiosis in poultry (Christaki et al., 2012)Hence the intake of EOs with feed can act
as best alternative to anticoccidialdrugs for treating avian coccidiosis(Bozkurt et al., 2013;
McCloud, 2010; Tsinas et al., 2011)
Plants validated for anthelmintic activity includes A. Indica (Iqbal et al., 2010) as well
T.ammi(Lateef et al., 2006).

Trachyspermumammicommnely known as “Ajwain” is mostly used old as well advancded medical

value with potent drug ,antimicrobial, antifungal, nematicidal, anthelmintic, carminative having,
antiulcer, antitussive, hepatoprotective, bronchodilator, antiviral and diuretic effects mentioned by
Persian clininicians. Ajwain is well known for its brownish color of essential oil have active part
“thymol” and “carvacrol” that countracts with parasite energy mechanism ,disrupts energy system
utilized and potentiate ATPase and cholinergic process leads expel out parasite from body.
Nemacidal activity of ajwain essential oil shown due to thymol as well carvacrol.(Zarshenas et al.,
2013)

Most researchers studied the effect major constituents of Trachyspermumammi that are Thymol
and Carvacolwere intakan in broilers chickens which were infected already at 28th day with
spourulatedovocyst of E. acervulina. It was concluded that medication of Thymol as wellCarvacol
to infected broilers chickens enhanced the integrity of intestine by reducing coccidiosis(Greathead
and Kamel, 2006)
It was reported by the reserchers that terpenes that are that are the active ingridients in the essential
oils of Eucalyptus and peppermint oils are proved as growth promoters with coccidiostat effect in
Eimeria species infection in broilers chickens and challenges as improvd feed conversion ratio,
lower intestinal lesion, decrease in mortality level, drop in ovocyst count in dung (Barbour et al.,
2015)Herbal complex of Azadirachtaindica, Nicotinatabacum, Calotropisprocera and

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Trachyspermumammiconcentration (2g, 4g, 6g) given in water to broilers chickens infected with
coccidiosis especially E.tenella and same medication was compared with amprolium treated group
of chickens concluded that herbal mixture proved to be more effective treatment against E.
tenellainfectionwhen given orally to chicken infected with Eimeria infection at a dose rate of 2-6g
once proved to be increased weight gain, decrese mortality rate, decrease lesion scoring, increase
Feed conversion ratio (Zaman et al., 2012).

Thymol as major component of ajwain(Khajeh et al., 2004)act as allosteric modulator for the
insects gamma-aminobutyric acid(GABA) receptor(González-Coloma et al., 2002)and hence it
may act as ascaricidal potential due to its active components that are present in seeds of
Trachyspermumammi(Lateef et al., 2006)

Trachyspermumammi belong to Apiaceae family plants having important constituents are the
Thymol and carvacrol. The seeds of Trachyspermumammi plant having numerious medicinal
properties like antispasmodic, antitussive, cardiovascular, respiratory and liver protectant,
gastrointestinal, urogenital, antimicrobial, antiparasitic effects (Boskabady et al., 2014). So this
plant would be a therapeutic value in treating various disorders.

Therefore further clinical trials and study is needed to investigate the anticoccidial potential of
essential oils of Trachyspermum ammi against antiparasitic especially for Eimeria infections in
poultry industry.

3. Materials and Methods

3.1. Plant materials
The Essential oil of Trachyspermum ammi (oil of Seed, leaf, root or Whole plant oil???PLZ
Mention here) will be purchased from registered company name write here??? local market of
Faisalabad (Pakistan), identified as well analysized by a botanist by storing in the herbarium of
Department of Botany, Univerisity of Agriculture Faisalabad, Pakistan.

3.2. Collection of coccidialoocysts

The suspected chicken guts infected naturally with Coccidiosis will be collected together from
various poultry shops From Faisalabad market. Opened the guts and contents examined by
microscope that obtained from guts. These suspected contents were separately placed in dedicators

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solutions 25% sodium hypochlorite solution at a ratio of 4:1 for 25-30 minutes to remove the
debris. To expel out water chemical contents round about three to four times water will be added
to the desiccators solution to obtain sediment in it. Next oocyst of Coccidiosis will be obtained by
following extraction technique that detailed described by (Ryley et al., 1976)

3.3. Sporulation of Oocysts

Positive samples will be placed in solution of potassium dichromate 2.5% having thickness upto
6mm in petri dishes. To ensure the regular supply of oxygen petri dishes will be covered partially
for upto 48 hours at a temperature of 39-35 C for incubation at a humidity level giving 60-80 %.
(Ryley et al., 1976). Continuous stirring of the petri dishes contents will be done at regular interval
of each four hours to give proper oxygen.DuringLevel of humidity will be maintained upto 60-80
percent by putting water in two separate petri dishes in the incubator during a process of
sporulation. By using inverted microscope the oocyst sporulation is verified by picking a drop of
contents from the petri dishes for examination after sporulation process completion.

3.4. Isolation of the sporulated oocysts

The separation of sporulated oocyst will be done by a process of zinc floatation technique (Ryley
et al., 1976). The equall quantity of zinc sulphate solution and oocyst sporulation solution will be
mixed and centrifuge at a rate of 1500 revolution per minute for ten minutes.
Sporulatedoocystcontaing supernatant solution will be sucked up by using pippete and separated
the solution in the jar. The water quantity is mixed in the supernatant at a ratio of 1:5 and kept the
whole solution overnight undisturbed. From pipetting system sporulatedoocyst will be collected
in the bottom layer by removing all the water upto one inch above the jar through the suction
process. Centrifugation of the bottom layer containing sporulatedoocyst will be done at a rate of
1500 rpm for 10 minutes by discarding supernatant round about third-forth portion.The washing
will be done of the one fourth portion of solution having sporulatedoocyst at two to three times by
using phosphate buffered saline solution. After washing counting of the sporulatedoocyst will be
started with the help of McMaster chamber (Lapolt et al., 1986) The minimum required dose of
sporulatedoocyst will be preserved with phosphate buffered saline solution that should be 60,000
per ml.

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3.5. Experimental Design ( should be decided)
????/???? full Deigns. groups, dose of EO? ? mention here after finalizing
In vitro study design??
In vivo study design I suggest in vivo study is enough for M.phil degree level PLZ ALSO
COCNSULT it WITH RAO SB
3.6. Parameters
The following parameters data will be recorded
3.6.1. Weight gain
The weighing of all chicks under trial of each groups will be measured properly at inaoculation
day and weighing again until at 42-45 day during whole the experimental process. The weight gain
in percentage of chicks under observation will be examined.
3.6.2. Feed conversion ratio

The feed ratio will be designed by the formula

Ratio of feed conversion = Mean of feed utilized in grams /Mean of weight gain in grams

3.6.3. Lesion Scoring

The process of lesion scoring process will be assessed at day seventh after inoculation with the
help of macroscopic examination. The scoring will be monitored from 0 to 4 by using Lesion
scoring procedure (Johnson and Reid, 1970)
3.6.4. Oocyst Scoring
From microscopic examination of scrapings from chicks intestine that will be slaughtered to
perform lesion scoring at day seventh of post inaculation the oocyst scoring will be done from 0
to 5 (Hilbrich, 1978)

3.6.5. Fecal Scoring

From day 3rd to 7th after inoculation a fecal scoring will be performed from 1 to 5 : normal faeces
represent a score 1, and severe diarrohea with adequate quantity of blood mixed in it indicate the
score number 5 (Youn et al., 1993)
3.6.5. Mortality

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The mortality rate of the chicks will be monitored as well post mortem examination will be
operated to evaluate the reason of mortality of chicks at the death time.

3.6.6. Hematological Parameters

For blood collection chickens will be slaughtered on the 7th day after inoculation from each
experimental unit. By using microhaematocrit method the values of pack cell volume and will be
measured properly. From Sahlis apparatus concentration of haemoglobin will be determined. The
counting of leukocyte as well erythrocyte will be performed with the help of Haemocytometer
chamber at a dilution rate of 1:200 of blood dilution in the Natt and Herrick solution (Natt and
Herrick, 1952).

3.6.7. Serum Chemistry

The serum chemistry of each group will be performed from day 7th of post inoculation by the help
of commercially available kits in the market.
3.7.8. Statistical Analysis
The statistical significance will be monitored by applying the one way analysis of variance
(ANOVA) and Duncan s tests of multiple range. From Chi square test the percentage of the data
recorded as well percentage solvability rate will be evaluated. (Add refrence here???).

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Signatures

Name of student : Asghar Abbas ___________

Supervisor : Dr. Rao Zahid Abbas ___________

SUPERVISORY COMMITTEE:
1. Dr. Rao Zahid Abbas (Chairman) ______________________
2. Dr.Zia ud Din Sindhu(Member) ______________________
3. Dr. Muhammad Anjum Zia (Member) ______________________

FORWARDED:

CHAIRMAN,
Department of Parasitology,
University of Agriculture,
Faisalabad.

SCRUTINY COMMITTEE:
Prof. Dr. Zargham Khan (Convener) ______________________
Dr. Ijaz Ahmad (Member) ______________________
Dr. Faqir Muhammad (Member) ______________________
Dr. M. Shahid Mahmood (Member) ______________________
Dr. Rao Zahid Abbas (Member) ______________________

REVIED AND WITNESSED:

DEAN, DIRECOR,
Faculty of Veterinary Science, Graduate studies,
University of Agriculture, Faisalabad. University of Agriculture,Faisalabad.

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