Application for permission for Animal Experiments

Application to be submitted to send either to the CPCSEA (address in form A

above) or Institutional Animal Ethics Committee (IAEC).

Para A

*1. Name and address of establishment

*2. Registration number and date of registration

3. Name, address and registration number of breeder from whom animals acquired (or to
be acquired) for experiments mentioned in parts B and C

CCSHAU Farm (Dept. of Animal Breeding). If not available, than from local
farmer.

4. Place where the animals are presently kept (or proposed to be kept)

Animal House, Dept. of Vety. Pathology, COVS, CCSHAU, Hisar.

5. Place where the experiment is to be performed

Dept. of Vety. Pathology, COVS, CCSHAU, Hisar.

6. Date on which the experiment is to commence and duration of experiment

In month of March, 2009 or just after getting permission.
Duration of Experiment: Approx. 2-3 months

Duration of Experiment:
(The appropriate protocols form for the research proposal - Part B in the case of
experiments using animals other than non human primates, part C for use of non-
human primates – to be duly filled in, signed and appended to this form)

Signature
Name and Designation of Chief Investigator
Date

Place
_______________________________________________________________________
_
*Applicable only for application to be submitted to CPCSEA
Part B

Protocol form for research proposal to be submitted to the Committee/Institutional

Animal Ethics Committee, for new experiments or extensions of ongoing
experiments using animals other than non-human primates.

1. Project Title: Pathobiological and Immunological Studies on

Chlorpyrifos Toxicity and its Interaction with
Salmonella infection in Broiler Chickens

2. Chief Investigator:

a. Name: Dr. R.P. Gupta

b. Designation: Professor

c. Deptt./Div./Lab.: Veterinary Pathology

d. Telephone number: 289254

3. List of names of all individuals authorized to conduct procedures under this

proposal

1. Dr. R.P. Gupta

2. Dr. Vikas Nehra (Ph,D. student)
3. Dr. S.K. Mishra

4. Funding Source: State Scheme Plan

5. Duration of the project:
a. Number of months: Approx. 2-3 months
b. Date of initiation: In March, 2009 or just after getting permission
c. Date of completion: October, 2009 (Including laboratory work)
d. 6. If date by which approval is needed is less than six weeks from dates of
submission, justification for the same:

Student wants to start at the earliest.

7. Study Objectives {The Aims of Study (and why they are important) to be
explained briefly using non-technical terms as far as possible}
Pathobiological and Immunological Studies on Chlorpyrifos Toxicity and its
Interaction with Salmonella infection in Broiler Chickens. Haematological,
Biochemical, Immunological and Pathological studies will be undertaken.
 8. Animal required:
a. Species: Broiler chicken
b. Age/Weight/Size: Day-old
c. Gender: Either sex
c. Numbers to be used (Year-wise break-ups and total figures needed to be given):
260
d. Number of days each animal will be housed: Approx. 8-9 weeks
9. Rationale for animal usage:
a. Why is animal usage necessary for these studies?
Use of birds is necessary to investigate Pathobiological and Immunological
Studies on Chlorpyrifos Toxicity and its Interaction with Salmonella infection in
Broiler Chickens.
b. Why are the particular species selected required?

Organophosphate insecticides have been reported to cause immunosuppression in poultry and

thus their exposure may increase susceptibility to opportunistic organisms. So in vivo study on
broiler chickens is required.
c. Why is the estimated number of animals essential?
Plan of the work is enclosed which includes experimental design.
d. Similar experiments conducted in the past. If so, the number of animals used and results
obtained in brief
No
e. If yes, why new experiment is required? N.A.

f. Have similar experiment(s) been made by any other organization/agency? If so, their
results in your knowledge

In my knowledge, no.

10. Description of procedures to be used

(List and description of all invasive and potentially stressful non-invasive procedures that animals will be subjected to in the course of
the experiment, indication of the frequency for all procedures where appropriate. The following specific issues are also to be addressed
when relevant-injections (substances, doses, sites and volumes), blood withdrawal (volumes and sites), radiation (dosage and
schedules), all anaesthetics and/or analgesics (dosage and routes), mechanical methods of restraint, animal identification methods,
methods of non-survival surgical procedures and experimental endpoint criteria (required when pathological changes are expected to be
caused).

Tentative protocol for the research project is enclosed. Withdrawal of blood from heart
at regular intervals and sacrificing of 5 birds at each interval from each subgroup.
11. Does the protocol prohibit use of anaesthetic or analgesic for the conduct of painful
procedures (any which cause more pain than that associated with routine injection, or
blood withdrawal)?

If yes, explanation and justification: Yes, Anaesthetics/and analgesic drugs will

effect the results.

12. Will survival surgery be done? If yes, the following to be described: No

a. List and description of all such surgical procedures (including methods of asepsis):

N.A.

b. Names, qualifications and experience levels of operators:

N.A.

c. Description of post-operative care: N.A.

d. Justification if major survival surgery is to be performed more than once on a single

individual animal:

N.A.

13. Methods of disposal post-experimentation:

Rehabilitation/Euthanasia (in case of euthanasia, justification for not undertaking

rehabilitation and drug dosage and route for anaesthesia, where appropriate, as well as
methods of carcass disposal):

Naturally dead and sacrificed birds will be disposed off properly away from
post-mortem room. Birds will be euthanized by dislocation of atlanto-occipital joint.
14. Animal transportation methods if extra-institutional transport is envisaged:

Through well ventilated card board boxes.

15. Use of hazardous agents (use of recombinant DNA-based agents or potential human pathogens
requires documented approval of the Institutional Biosafety Committee (IBC). For each category, the
agents and the biosafety level required, appropriate measures and the mode of disposal of
contaminated food, animal, wastes and carcasses must be identified).

No
e. Any other (give name): -
Copy of IBC approval to be attached in case hazardous agents are used.
d. Recombinant DNA: -
Investigator’s declaration
1. I certify that I have determined that the research proposal herein is not unnecessarily duplicative of
previously reported research.
2. I certify that all individuals working on this proposal, and experimenting on the animals, have been
trained in animal handling procedures.
3. For procedures listed a. Radionuclides: -

b. Biological Agents: -

c. Hazardous chemicals or drugs: -

under item 11, I certify that I have reviewed the pertinent scientific literature and have found no valid
alternative to any procedure described herein which may cause less pain or distress.
4. I will obtain approval from the IAEC/CPCSEA before initiating any significant changes in this study.
5. Certified that performance of experiment will be initiated only upon review and approval of scientific
intent by appropriate expert body {institutional scientific Advisory Committee/funding agency/other
body (to be named)}.
6. Institutional Biosafety Committee’s (IBC) certification of review and concurrence will be taken
(Required for studies utilizing DNA agents of human pathogens).
7. I shall maintain all the records as per form (Form D).

Signature
Date: Name of Investigator

______________________________________________________________________________________

(for IAEC/CPCSEA usage)

Proposal number
Date first received
Date received after modification (if any)
Date received after second modification (if any)
Approval date
Expiry date
Name of IAEC/CPCSEA Chairperson
Date Signature
 Department of Veterinary Pathology

6. Title of the Project: Pathobiological and Immunological Studies on

Chlorpyrifos Toxicity and its Interaction with
Salmonella infection in Broiler Chickens.

TECHNICAL PROGRAMME OF WORK

Experimental birds:
Two hundred and sixty, day old broiler chicks will be reared in the departmental animal
house under strict hygienic conditions providing feed and water ad libitum. From this, thirty birds
will be used for calculation of absolute lethal dose (ALD50).
Determination of ALD50:
A pilot experiment on thirty birds will be conducted to calculate absolute lethal dose
(ALD50) of chlorpyrifos as per the method of Reed and Muench (1938).
Experimental design:
Two hundred and thirty (230) broiler chicks at the age of 14 days will be divided
randomly into three groups (group A, B and C) of 80, 80 and 70 chicks respectively in each
group. All the birds of group A will be given feed mixed with 1/10th of ALD50 of Chlorpyrifos and
that of group B will be given feed mixed with 1/20th of ALD50 of Chlorpyrifos. Each bird of
group C will receive plain feed. At the age of 21 days these three groups will be further divided
into two subgroups (group A into A1 and A2, group B into B1 and B2 and group C into C1 and C2)
of 40 birds each in subgroup A1, A2, B1, B2 and C2, group C1 will be having 30 birds. Each bird of
group A2, B2 and C2 will be injected with infective dose (ID50) of Salmonella infection. Blood and
Serum samples will be collected from five birds of each group on 0th, 3rd, 7th, 14th, 21st and 28th day
post infection for haematological, biochemical and immunological studies. After collection of
blood and serum, these birds will be sacrificed. They will be subjected to thorough post-mortem
examination. Representative pieces of liver, heart, lungs, brain, kidneys, intestine, spleen, thymus
and Bursa of Fabricius will be collected in 10 per cent buffered formalin for histopathological
studies.
Observations:

1. Body weight gain: Birds will be weighed on days 14, 17, 21, 28, 35, and 42 of the
age.

2. Clinical signs and mortality: Birds will be closely observed daily for clinical signs
and mortality, if any.

3. Haematological studies: Blood samples collected at different intervals will be

analysed for the following parameters.
a. Haemoglobin concentration (Hb).
b. Packed cell volume (PCV).
c. Total erythrocyte count (TEC).
d. Total leucocyte count (TLC).
e. Differential leucocyte count (DLC).
f. Mean corpuscular volume (MCV) and Mean corpuscular haemoglobin
concentration (MCHC).
4. Biochemical studies: Serum samples collected at different intervals will be analysed
for the following parameters.
a. Total protein and Albumin concentrations.
b. Serum sodium and potassium concentration.
c. Serum Aspartate transaminase activity.
d. Serum Alanine transaminase activity.
e. Serum Creatine phosphokinase activity.
f. Serum Alkaline phosphatase activity.
g. Serum Acetylcholine esterase activity.

5. Viable bacterial cell count in Salmonella infection: Viable bacterial cells will be
counted in broiler chicken at 0, 3, 7, 14, 21 & 28 days post infection.

6. Pathological studies
a. Gross changes: Gross lesions, if any, will be recorded during post mortem
examination of the birds sacrificed or died naturally.
b. Histopathological changes: The formalin fixed tissues will be processed for
paraffin embedding technique. The sections will be stained with haematoxylin
and eosin for histopathological interpretation (Luna, 1968).

7. Immunological studies: Blood serum will be collected at 0, 3, 7, 14, 21 & 28 days

post infection from at least 5 birds and immune response will be studied as follows:-
A. Study of humoral mediated immune response (HMI):- Humoral immune response
will be studied against Salmonella infection treated with chlorpyrifos by indirect
ELISA.
B. Study of cell mediated immune response (CMI):-
a. The CMI will be studied by lymphocyte proliferation assay at each of the six time
intervals as in humoral immune response. The birds will be bled and their
lymphocyte will be separated by Ficoll-Hypaque. CMI will be studied for Salmonella
infection treated with chlorpyrifos and mitogen induced lymphoproliferation
response by MTT based lymphoproliferative assay.
b. The population of different T-cell markers i.e. CD4+, CD8+ will be analyzed using
monoclonal antibodies (mAbs) against these T-cell markers by flow
cytometry/immunoperoxidase technique as per the method of Sharma et al. (1990).