standardization of enzyme linked immunosorbent assay for the detection of antibodies to argas ...

139 Pages·2017·8.75 MB·English

STANDARDIZATION OF ENZYME LINKED IMMUNOSORBENT
ASSAY FOR THE DETECTION OF ANTIBODIES TO ARGAS
PERSICUS (OKEN, 1818) INFESTATION IN FOWL

By
POTTABATHULA MALLESH
B.V. Sc. & A.H
I.D No: RVM/2014-45

MASTER OF VETERINARY SCIENCE
(VETERINARY PARASITOLOGY)

DEPARTMENT OF VETERINARY PARASITOLOGY
COLLEGE OF VETERINARY SCIENCE
P.V. NARSIMHA RAO TELANGANA VETERINARY UNIVERSITY
RAJENDRANAGAR, HYDERABAD-500 030

FEBRUARY, 2017
STANDARDIZATION OF ENZYME LINKED IMMUNOSORBENT
ASSAY FOR THE DETECTION OF ANTIBODIES TO ARGAS
PERSICUS (OKEN, 1818) INFESTATION IN FOWL

By
POTTABATHULA MALLESH
B.V. Sc. & A.H
I.D No: RVM/2014-45

THESIS SUBMITTED TO
P.V. NARSIMHA RAO TELANGANA VETERINARY UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF

MASTER OF VETERINARY SCIENCE
(VETERINARY PARASITOLOGY)

DEPARTMENT OF VETERINARY PARASITOLOGY
COLLEGE OF VETERINARY SCIENCE
P.V. NARSIMHA RAO TELANGANA VETERINARY UNIVERSITY
RAJENDRANAGAR, HYDERABAD-500 030

FEBRUARY, 2017
CERTIFICATE

I certify that, POTTABATHULA MALLESH has satisfactorily prosecuted the
course of research and the thesis entitled “STANDARDIZATION OF ENZYME LINKED
IMMUNOSORBENT ASSAY FOR THE DETECTION OF ANTIBODIES TO ARGAS
PERSICUS (OKEN, 1818) INFESTATION IN FOWL” submitted is the result of original
research work and is of sufficiently high standard to warrant its presentation to the
examination.
I also certify that the thesis or part thereof has not been previously submitted by
him for a degree of any University.

Date: (M.UDAYA KUMAR)
Place: Hyderabad Major Advisor
DECLARATION
I, POTTABATHULA MALLESH hereby declare that the thesis entitled
“STANDARDIZATION OF ENZYME LINKED IMMUNOSORBENT ASSAY FOR
THE DETECTION OF ANTIBODIES TO ARGAS PERSICUS (OKEN, 1818)
INFESTATION IN FOWL” submitted to P.V.NARSIMHA RAO TELANGANA
VETERINARY UNIVERSITY for the degree of MASTER OF VETERINARY
SCIENCE is the result of original research work done by me. It is further declared that the
thesis or any part thereof has not been published earlier in any manner.

Date:
Place: Hyderabad (POTTABATHULA MALLESH)
CERTIFICATE
This is to certify that the thesis entitled “STANDARDIZATION OF ENZYME
LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF ANTIBODIES
TO ARGAS PERSICUS (OKEN, 1818) INFESTATION IN FOWL” submitted in partial
fulfillment of the requirements for the degree of MASTER OF VETERINARY SCIENCE
of the P.V. NARSIMHA RAO TELANGANA VETERINARY UNIVERSITY,
HYDERABAD is a record of the bonafide research work carried out by POTTABATHULA
MALLESH under my guidance and supervision. The subject of the thesis has been approved
by the student’s advisory committee.
No part of the thesis has been submitted for any other degree or diploma. The
published part has been fully acknowledged. All assistance and help received during the
course of the investigations have been duly acknowledged by the author of the thesis.

(M.UDAYA KUMAR)
Chairman of the Advisory Committee
Committee

Thesis approved by the Student’s Advisory Committee.

Chairman : Dr. MANCHUKONDA UDAYA KUMAR
Professor & University Head
Dept. of Veterinary Parasitology
College of Veterinary Science
Korutla, JagitIal Dt.- 505326. (Telangana)

Member: Dr.G.S.SREENIVASA MURTHY
Associate Professor & Head
Dept. of Veterinary Parasitology
College of Veterinary Science
Rajendranagar, Hyderabad- 30. (Telangana)

Member: Dr. M. LAKSHMAN
Professor
Dept. of Veterinary Pathology
College of Veterinary Science
Rajendranagar, Hyderabad-30. (Telangana)
ACKNOWLEDGEMENT
First and definitely foremost, I would like to express my sincere appreciation and
gratitude to my major guide Dr. Manchukonda Udaya Kumar, Professor and University
Head, Department of Veterinary Parasitology, College of Veterinary Science, Korutla for his
kind support. His high standards, careful planning and inspiring suggestions have allowed
this research work to be successful. The concept and original design of this project came
from him which is a very important contribution.
I am also indebted to Dr. G. S. Sreenivasa Murthy, Associate Professor & Head,
Department of Veterinary Parasitology, College of Veterinary Science, Hyderabad, who has
been a constant source of encouragement, enthusiasm, instruction, suggestions, guidance
and affectionate feelings at all stages of research work, not only during this thesis project
but also during my Master program.
I would also like to thank to Dr. M. Lakshman, Professor & officer in charge of
Ruska labs, Department of Veterinary Pathology, College of Veterinary Science, Hyderabad
member of the Advisory committee for his help, professionalism, valuable instruction,
suggestions and affectionate feelings in technical script in scanning electron microscopy.
I am extremely thankful to Dr. I. Balasundaram, Veterinary Assistant Surgeon,
Siddipet for their help during sample collection essential for my research work.
I am verymuch thankful to Dr. Sudhakar Kommu and Dr. G. Rajeshwari Contract
Teaching Faculty, Department of Veterinary Parasitology for their help during course of
work.
I am extremely thankful to Dr. S. Vanisri, Senior scientist and Dr. Madhu, Research
Associate, Institute of Biotechnology for their help during my research work.
I am very much thankful to Dr. M. Hanumanth Rao, Assistant professor, Department
of Poultry Science for their help during blood collection.
I am very much thankful to Dr. Sameta Ramesh, Assistant professor, Department of
Parasitology for their help during my course work.
I gratefully acknowledge the help, encouragement and understanding of my friend
Dr. Lubna which have provided good and smooth basis for my M.V.Sc tenure.
I am verymuch thanks to my friends Dr. Vamshi, Dr. Ravi Teja, Dr. Sampath, Dr.
Bala Krishna and Dr. Vijay for their cooperation during my M.V.Sc tenure.
I am very glad to acknowledge the encouragement, support and help provided by my
senior Dr. Rasmita Panda and thanks to my juniors Dr. Nagendra Babu and Dr. Deepthi
for their cooperation during the entire research work.
It gives me immense pleasure to express my profound sense of gratitude and sincere
thanks to L. Atchamamba, Assistant Professor, Rajesh and Kishore of College Library for
their help in providing the necessary facilities and to carryout research work.
I acknowledge the help and assistance of the non-teaching staff Kishan, Yadamma
and Bhagyamma of Department of Veterinary Parasitology.
I must express my very profound gratitude to my father Sri. Ramulu, mother Smt.
Parvathamma and my sister Sharadha for providing me with unfailing support and
continuous encouragement throughout my years of study and through the process of
researching and writing this thesis. This accomplishment would not have been possible
without them.
I express my gratefulness to the Lord Sree Mallikarjuna who has blessed me with life,
time, energy and enabled me to pursue my higher education to complete the research work
and the thesis for the degree of Master of Veterinary Science in Parasitology.
Finally, my greatest regards to the Almighty for bestowing upon me the courage to
face the complexities of life and complete this project successfully.
Last but not least, I would like to thank P V Narsimha Rao Telangana Veterinary
University for giving financial support in the form of stipend to complete the course.

Pottabathula Mallesh
Name of the Author : POTTABATHULA MALLESH
Title of the Thesis : STANDARDIZATION OF ENZYME LINKED
IMMUNOSORBENT ASSAY FOR THE
DETECTION OF ANTIBODIES TO ARGAS
PERSICUS (OKEN, 1818) INFESTATION IN
FOWL
Degree to which : MASTER OF VETERINARY SCIENCE
it is submitted
Faculty : VETERINARY SCIENCE
Discipline : VETERINARY PARASITOLOGY
Major Advisor : Dr. MANCHUKONDA UDAYA KUMAR
University : P.V. NARSIMHA RAO TELANGANA
VETERINARY UNIVERSITY, HYDERABAD-30,
TELANGANA
Year of Submission : 2017
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ABSTRACT
The fowl tick, Argas persicus (Oken, 1818) is a soft tick of domestic and certain wild
birds in parts of Asia, Europe, Africa and Australia. An ELISA was standardized to detect
antibodies to Argas persicus infestation in chickens and the sensitivity and specificities were
determined.
The morphological characters of Argas persicus were confirmed by light microscopy
and scanning electron microscopy (SEM) prior to ELISA. Macroscopically, engorged ticks
were slaty blue in colour and unengorged ticks were yellowish brown in colour. The adult
ticks were oval in shape, narrower anteriorly than posteriorly. Light microscopy revealed
semi-circular genital aperture in male where as female tick was identified by horizontal
genital aperture. Eggs were brownish in colour and spherical in shape. The mouthparts in
larva were situated anteriorly. Whereas, SEM studies showed spherical and oval eggs. The
spherical eggs were of 418 ± 11.15 µm in diameter. The mean length and width of oval
shaped eggs were 399.5 ± 0.50 µm and 452.5 ± 9.5 µm.
The larva of Argas persicus showed three pairs of legs and anus at posterior third of
idiosoma. The anus of Argas persicus larva (APL) showed vertical slit guarded by two leaf
like valves. The tarsus of first pair of legs of APL showed Haller’s organ with anterior pit
and posterior capsule. The anterior pit was circular and contained seven sensilla, while the
posterior capsule had twenty dorsal apertures. Dorsal view of APL showed centrally located
dorsal plate and capitulum located near the anterior margin of ventral aspect. Spherical
tubercles and elongate tubercles were seen on the dorsal plate. Ventral view of mouthparts
consisted of four longitudinal rows of posteriorly-oriented conical denticles on hypostome.
The mouthparts of adult Argas persicus in SEM showed palps, chelicera, hypostome,
mechano-sensilla and basis capitulum. The anterior margin of genital aperture of female was
having numerous sensilla. In female, the anus was surrounded by a wide oval perianal plate
of hard cuticle.
The paired salivary glands of fully engorged male and female ticks were visualized
by staining with Methyl green pyronin, Giemsa and FAAL which consisted of two types of
alveoli viz., oval or pear shaped type I alveoli with a peripheral and central zone and the
circular or oval type II alveoli consisting of three types of cells surrounding a central lumen.
These cells were triangular in outline with the base of triangle towards the alveolar periphery.
The antigen was prepared by homogenizing the salivary glands in PBS (pH 7.4) in
glass homogenizer followed by sonication at 20 Watts of 6 cycles, each lasting for 30 seconds
with an interval of 30 seconds in between. Then the suspension was centrifuged at 18,000 ×