STANDARD OPERATING PROCEDURE FOR ANIMAL  DISEASE OUTBREAKS IN INDIA

 

STANDARD OPERATING PROCEDURE FOR ANIMAL  DISEASE OUTBREAKS IN INDIA

 

Compiled & shared by-DR RAJESH KUMAR SINGH ,JAMSHEDPUR,JHARKHAND, INDIA, 9431309542,rajeshsinghvet@gmail.com

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An outbreak is the occurrence of cases of an illness, specific health related behavior or other event, clearly in excess of normal expectancy in a community in a specific time period. An outbreak is limited or localized to a village, town or closed institution. However, the magnitude could involve wider geographic areas, even beyond one district, thus called an epidemic (A dictionary of Epidemiology, 4th Edition, 2001, John M Last). In case of diseases which are under eradication or elimination phase; even a single case of such disease may be treated as an outbreak, e.g. Rinderpest. Rare but internationally important diseases, with high case fatality rate and with the potential of importation due to existence of conducive epidemiological conditions e.g. avian influenza etc. and in the end outbreaks of unknown diseases/syndromes.

The occurrence of more than the expected number of cases included in the scheduled list (Annexure-I) shall be the start of the outbreak and will warrant the initiation of control and containment measures immediately by the local Veterinary Officer with the help of his staff. The Joint Director/Chief Veterinary Officer (JD/CVO) of the respective district shall be informed immediately both telephonically and in writing. The complete details of the case/cases shall be maintained in the appropriate format (Annexure-II “CASE WISE OUTBREAK INFORMATION REPORT”). A separate sheet shall be filled for each case affected with the disease and a complete
record and follow up shall be maintained on a daily basis. This format will be maintained by the concerned  Veterinary  Officer  and  shall  be  preserved  at  his/her  Veterinary  Hospital.  The
animal/animals shall be maintained in a state of quarantine at the owner’s premises in a separate room and all other animals in contact shall be immediately removed to a separate location and the
owners, veterinary officers and other supporting staff shall also maintain sanitation protocols so that they do not serve as a nidus for the spread of infection to other areas. All possible effort will be
made to start therapeutic treatment in case of diseases where treatment exists and supportive treatment in case of diseases where treatment does not exist. In case vaccine against the disease is
available then immunization for the same shall be immediately started in a ring fashion. Ring fashion would mean that one or several teams shall start vaccinating from roughly a three kilometer
radius towards the center of the outbreak and another team shall start vaccination from roughly a six kilometer radius back towards the epicenter of the outbreak. At the end of the day a consolidated
outbreak   information   report   will   be   compiled   on   prescribed   format (Annexure-III,
“CONSOLIDATED OUTBREAK INFORMATION REPORT”) and sent to the office of the JD/CVO by fax/email/telephonically.

 

Endemic Outbreak (Non-Zoonotic)

 

In case the number of animals affected is less than fifty (50) and the disease is non-zoonotic in nature (excluding Rinderpest) and limited to a single location or village the outbreak shall be classified as an endemic.

 

As soon as information about any disease in several animals is reported from an area the VO of the concerned hospital along with whatever staff he/she deems necessary shall immediately proceed to the affected area.

 

Depending on the nature of the disease and initial diagnosis biological samples from affected animals and the ones in contact with the affected animals shall be collected and sent for confirmatory diagnosis to the nearest DIO laboratory. The specimens/samples to be collected will depend on the suspected disease; a detailed suggestive list for the type of samples to be collected for various diseases is upended to these directions as Annexure-IV.

In case treatment for the disease is possible then therapy will be started immediately and in case treatment is not possible then supportive therapy will be initiated accordingly. The farmers will be counseled and educated about the disease process and precautions to be undertaken; the importance of quarantine, isolation, segregation, sanitation, disinfection, etc. will be explained to them in detail so that they also participate in the containment process proactively.

 

All details of the outbreak will be recorded on prescribed formats i.e. Annexure-II, III & IV and sent to designated officers as indicated in the SOP.

 

In case further assistance is sought by the leader of the first team the Joint JD/CVO of the district shall constitute a team of Veterinary Officers (VO), Livestock Extension Officers (LEO) and other support staff of the adjoining Hospitals and LEO Centers. The VO of the concerned block shall be the leader of the said team. On receipt of information the members of the said team shall immediately convene at the concerned Veterinary Hospital and shall proceed to the area of outbreak to initiate relief operations immediately.

 

3.0 Epidemic Outbreak (Non-Zoonotic)

In case the number of affected animals exceeds by more than fifty (50) and are spread over a wider area or number of villages the outbreak shall be treated as an epidemic. The information about the incidence  shall  then  also  be  sent  to  the  Additional  Director  of  the  concerned  division telephonically and in writing by post as well as by fax and/or telegram and email. The information to be sent shall also be on prescribed format (Annexure-III) so that in case the disease spreads to other areas, uniformity is maintained in the reporting system and compilation of the same is done in a systematic manner. The format shall be filled up in quadruplicate with the first copy to be sent to Additional Director ,the second copy to JD/CVO of the concerned district; the third copy to the Joint Director (Disease Control), AHD Directorate and the fourth copy is to be retained at the Veterinary Hospital for record.

The team upon reaching the site shall try and assess the gravity of the situation and collect information on the following points:

 

1. Whether the initial diagnosis of the disease seems to be correct/doubtful
2. Whether all the affected animals are showing similar consistent signs/varying signs
3. Whether the number reported to be suffering from said disease is as reported.
4. Whether they have been segregated and quarantined from the healthy animals.
5. Whether necessary samples have been collected and if so have they been collected as per
   recommendations  for  the  said  disease. (Annexure-IV  “LIST  OF  DISEASES  AND SAMPLES TO BE COLLECTED”)
6. Whether samples have been analyzed on the spot (Patient Side) / at the Veterinary Hospital / sent to appropriate laboratory.
7. Whether the samples have been sent by post / courier / special messenger.
8. A tentative situation report (SITREP) along with consolidated outbreak report (Annexure-
   III) shall be sent to the office of the Joint Director (District) with a copy to Additional Director (Divisional HQ) and Joint Director-Disease Control (HQ) on prescribed format.
   (Annexure-V “SITUATION REPORT”). This report shall be dispatched daily by the close of office hours every day. The Joint Director of the district shall make it a point to inform and keep the district administration updated on the progress of the disease.
9. The report shall indicate whether the disease is simply endemic or epidemic and whether it is zoonotic or not.
10. The type and scale of assistance required including manpower, materials, medications, surgicals, disposables, vaccines and any other materials. (A suggestive list is enclosed for
    reference as Annexure-VIII) must be included in the estimation.
11. Whether check posts have been set up or not. In case they have been set up then their list with their location, officer/s incharge and contact phone number/s.

In case the outbreak is classified as an epidemic but non zoonotic then a control room shall be established immediately at a location having facilities of communication possibly including phone, fax, internet and police/forest radio network. On receipt of information of an outbreak the Additional Director ,shall immediately proceed to the affected area and shall remain constantly in contact with the Joint Director of the district and Disease Control (HQ) and if the need arises then shall also keep the Director AHD informed. He/She shall also establish liaison with the district administration and shall be responsible to keep the Commissioner of the respective division informed regularly about the non zoonotic epidemic.

 

The Additional Director shall be the complete incharge of this control room and shall also set up his camp office at this site. He shall oversee the management of the entire operations; his orders in all matters shall be final. He shall be assisted in this task by the Joint Director of the district. This control room shall by far and large be at a place as near as possible to the epicenter of the epidemic. This control room shall be manned 24 hours by a senior Veterinary Officer who shall be incharge and shall be assisted by a team of secretarial staff and a Chief  Veterinary Pharmacist to maintain inventory and ensure distribution of medicines, vaccines and materials. The Joint Director (District) shall ensure mobilization of all possible help from the district administration and shall also provide all logistical support to the response team.

 

The JD/CVO (District) shall also be responsible for informing the district administration and the Joint Director-Disease Control (HQ) about the nature of the outbreak and the actual situation on ground. Who in-turn shall be responsible for informing the Director, Animal Husbandry about the gravity of the situation and the measures adopted to contain the disease outbreak. It shall be the final decision of the Director, Animal Husbandry to access the situation and then finally inform the departmental Secretary and other officers of the state government.

Another control room shall be established at the Directorate of Animal Husbandry, and shall also be manned round the clock by the Officers of the Directorate. They shall compile all information being received  from  the  field  and  send  regular  situation  reports (SITREP)  on  prescribed  format (Annexure-V) to the following Officers daily at the close of day:

1. P.S. to H’onble Minister of Animal Husbandry, state
2. P.S. to Chief Secretary, state
3. P.S. to Animal Husbandry Commissioner, Ministry of Animal Husbandry & Dairying,
   Government of India
4. P.S. to Principal Secretary & Commissioner, Forest and Rural Development
5. P.S. to  Secretary,  Animal  Husbandry,  Ministry  of  Animal  Husbandry  &  Dairying,
   Government of India.
6. Secretary, Animal Husbandry,
7. Secretary, Disaster Management, state
8. Secretary, Forest
9. Commissioner,

 

Zoonotic Epidemic

 

Incase the disease is tentatively diagnosed to be zoonotic then the chronology of events to be followed will more or less be the same except for the containment and disinfection procedures to be
followed.

 

1. First and foremost the owners of the affected animals will have to be taken into confidence and they shall be made to understand the gravity of the situation being faced.
2. Secondly the Chief Medical Officer of the district shall be informed immediately about the occurrence of the disease so that they can initiate action as per their protocols immediately since human lives are at stake. The people or the family members of the affected animals shall be immediately vaccinated in case a vaccine for the disease exists and all medical help shall be made available to them by the concerned department.
3. Serum and other necessary samples for the disease shall be drawn and sent to the referral laboratory for confirmatory diagnosis. The samples shall be drawn as per guidelines given in Annexure-V and send by special messenger to the designated diagnostic laboratory. In addition to this a second set of samples shall be sent to the National Center for Disease
   Control (NCDC), New Delhi for secondary confirmation since the nature of the suspected  disease is zoonotic.
4. In case the nature of the disease is such that treatment is possible then immediate treatment of the affected animals shall be initiated whereas in case no treatment exists then the  recommended procedure for culling and disposal shall be adopted.
5. In case vaccination for the said disease is available then ring vaccinations shall be immediately started in a manner already described.
6. Once official confirmation of the zoonotic disease is attained from the designated laboratory then immediate action shall be initiated on the following counts:

(A) The Director, Animal Husbandry will immediately inform the Secretary, AHD about all aspects of the disease including the causative agent; the morbidity and mortality  numbers; whether the disease is single or multiple species; steps already taken for the containment of the disease; status of the SOP already initiated; availability of veterinary medical supplies and all other materials and equipments as per Annexure- VI.

(B) The position of manpower in the affected area and the formation of additional response teams if required; the number of check posts setup. All roads, foot paths and other approach roads to and fro from the area should be covered so that movement of all animals and humans is monitored and/or stopped as the case may be.

(C) In case it is felt that the disease is assuming epidemic proportions then on the advice  of the Director AHD, and in case the Government sees reason then necessary notification shall be issued covering the district in full and the state in part or in full so that movement of the specified species of animals in question or the movement of several species may be restricted within the boundaries specified (intra district, inter-district, interstate). If the situation so desires then the all human and vehicular traffic movement to and fro from the area of the disease may/will also be stopped and covered by this notification.

(D) In case a notification to this effect is issued then a copy of the same shall be sent to  the P.S. to H’nible Minster of Animal Husbandry; P.S. Chief Secretary; P.S. to Principal Secretary & Commissioner, Forest and Rural Development; Secretary,
Department of Health & Family Welfare; Secretary, Department of Home; Director General  Police,  Secretary,  Department  of  Animal  Husbandry  &
Dairying-Government of India; Animal Husbandry Commissioner, Department of
Animal Husbandry & Daorying, Government of India; Director General, Remount
Veterinary Core; Director General, Indo Tibetan Border Police; Commandant,
Indian Military Academy and Secretaries and Directors of Animal Husbandry of
neighbouring states;.

(E) An immediate meeting of the following departments will be convened and shall be
organized by the Director Animal Husbandry as the Member-Secretary and will be
chaired by the Principal Secretary & Commissioner, Forest & Rural Development.
The following officers of various departments shall be the constituent members of
this meeting:

 

(i)          Principal Secretary & Commissioner, Forest & Rural Development

(ii)        Secretary Animal Husbandry & Dairying

(iii)       Secretary, Health & Family Welfare

(iv)       Secretary, Urban Development

(v)        Secretary, Revenue

(vi)       Secretary, Disaster Management

(vii)      Secretary, Public Works Department

(viii)     Secretary, Home

(ix)       Secretary, Panchayat Raj

(x)        Secretary, Forest

(xi)       Commissioner,

(xii)      Director General, Health & Family Welfare

(xiii)     Director, Urban Development

(xiv)     Chief Engineer, PWD

(xv)      Director General, Police

(xvi)     Director, Animal Husbandry

(F) Once the meeting is convened, detailed minutes of the meeting shall be prepared by
the office of the Director AHD and circulated to all concerned departments so that
action can be initiated as per decisions taken.

In case it is felt that a statement is to be issued to the general public and the media then the Secretary, Department of Animal Husbandry and/or the Secretary, Health & Family Welfare, shall issue the necessary statement on behalf of the Government.

 

NOTE: UNDER NO CIRCUMSTANCES WILL ANY STATEMENT BE GIVEN TO THE MEDIA
BY THE LOCAL OFFICERS, STAFF OF THE DEPARTMENT OF ANIMAL HUSBANDRY OR
ANY   OTHER   PERSONNEL   OF   ANY   DEPARTMENT   INVOLVED   IN   THE   DISEASE

CONTAINMENT PROCESS. IT WILL BE THE SOLE RESPONSIBILITY AND PURVIEW OF THE GOVERNMENT TO DO SO. THIS IS ESSENTIAL IN ORDER TO PREVENT PANIC AMONGST THE GENERAL PUBLIC.

 

(G) As far as the control of the disease amongst livestock is concerned the Additional
Director (HQ) shall be overall incharge of the entire outbreak management effort.
He shall be assisted in this function by the Joint Director (Disease Control) who
shall make an initial assessment of the situation and make necessary arrangement for
the movement of manpower, materials, medicines, vehicles and other equipment
required. Necessary orders for the same shall be prepared by him/her and after due
examination by the AD (HQ) shall be issued from the directorate.

(H) All officers and staff of the Directorate will be on a 24 hour standby. The Veterinary
Officers-I posted at the Directorate shall be sharing the following responsibilities
and shall take all necessary orders from JD(DC) and report to him/her directly:

 

(i)         Veterinary  Officer    (Disease  Control  &  Disaster  Management)        –

Logistics (arrangement of materials, medicines, vaccines, etc.)

(ii)        Veterinary Officer (Planning) – Transport Officer.

(iii)       Veterinary Officer   (Livestock)  – Compilation of outbreak data and

technical backstopping to VOs in the field.

(iv)       Veterinary Officer (MIS) – Documentation & Reporting.

(v)        Veterinary Officer    (Reproduction)  – Establishment     (composition of

RRTs, assigning of duties, etc.)

 

(I)  The number and size of the Rapid Response Teams (RRTs) to be formed shall
depend on the intensity and extent of the disease; geographical area involved; the
number of animals affected and whether any human cases have been reported or not.
The RRTs formed shall consist of one Senior Veterinary Officer, one Junior
Veterinary Officer, two Livestock Extension Officers and two fourth class staff
members. If need is felt then one Medical Officer shall also be a member of this
RRT. The decision to this effect shall be taken by the Department of Health &
Family Welfare after the assessment of the situation on the ground. If need is felt the
latter can also take up the issue of human health separately at their level and as per
their disease protocols and operating procedures.

(J) The first batch of RRTs shall be formed from the neighbouring districts and as
required RRTs shall be initially formed from the division where the outbreak has
occurred and secondly if the need arises from the districts of the other division. It is
imperative that all RRTs shall remain on duty for a fixed period of three weeks
thereafter they shall be replaced by a fresh team.

(K) The members of the RRTs shall take all necessary precautions with regard to their
personal health and shall strictly observe recommended procedure along with the
use of PPEs so that they do not accidentally become affected by that disease. In case
a vaccination for the disease in question is possible then all personnel shall be
vaccinated prior to departure for the affected area. In addition on the advice of the
Medical Health Department all necessary precautions as well as any preventive
medications if available will be made available to members of the RRTs.

(L) It shall be the responsibility of the Joint Director (Respective Districts) to arrange
for transportation of these RRTs from their districts to the area of duty either by
departmental vehicle or by hired vehicle. In no way shall these RRTs be ordered by
him/her to move by public transport as this will take time in reaching their
destination. On receipt of information all RRTs shall reach the control room of the
affected area within 24 hours from the receipt of their orders.

(M) As soon as they reach the control room they shall be issued with a set of instructions
to be followed; area in which they shall be working; complete kit consisting of
medicines, vaccines, surgicals, PPE kits, etc. and any other materials.

(N) Each  RRT  will  maintain  a  case  wise  outbreak  report  on  prescribed  format
(Annexure-II) [In case of poultry case wise report would mean household/farm wise
information]. Each RRT will ensure that by the end of the day (5.00 pm) they shall
send a copy of the consolidated report (Annexure-III) on prescribed format to the
local control room.

(O) On receiving the consolidated reports from all the RRTs in the field, the local
control room shall formulate a situation report (SITREP) on prescribed format
(Annexure-V) and send it by fax/email to the control room at the Directorate for
further consolidation.

(P) In case the disease is occurring in several locations of the state then consolidation of
all the SITREPS shall be done at the control room in the Directorate.

(Q) After consolidation of all SITREPS  from various locations a final situation report
shall be prepared and a copy of the same shall be forwarded to the following officers
of the state:

 

1. P.S. to H’onble Minister of Animal Husbandry, state
2. P.S. to Chief Secretary, state

iii.        P.S.  to  Animal  Husbandry  Commissioner,  Ministry  of  Animal

Husbandry & Dairying,   Government of India

1. P.S. to  Principal  Secretary  &  Commissioner,  Forest  and  Rural

Development

1. P.S. to Secretary, Animal Husbandry, Ministry of Animal Husbandry

& Dairying,   Government of India.

1. Secretary, Animal Husbandry, state

vii.       Secretary, Disaster Management, state

viii.      Commissioner,

1. Secretary, Health & Family Welfare
2. Secretary, Urban Development
3. Secretary, Revenue

xii.       Secretary, Disaster Management

xiii.      Secretary, Public Works Department

xiv.      Secretary, Home

1. Secretary, Panchayat Raj

xvi.      Director General, Health & Family Welfare

xvii.     Director, Urban Development

xviii.    Chief Engineer, PWD

xix.      Director General, Police

1. Director, Animal Husbandry

(R) Each RRT shall remain in the area of their operation for a minimum period of three
weeks or in case the disease gets controlled earlier. This condition has been put in
place since more the numbers of relief personnel that exit from the area of outbreak
the higher is the chance of spread of the disease to other areas. This time period shall
also not exceed above three weeks as the personnel working in these areas shall get
stressed out by such time hence a replacement RRT shall be provided.

(S) When exiting the area of their operation all members of the RRTs (including
clothings, shoes, personal articles and vehicles) shall be thoroughly disinfected and
sanitized as per prescribed protocol.

(T) Depending on the ground situation one or several emergency RRTs shall also be
formed for performing emergency duties during the night. These RRTs shall also act
as reserve teams in case their services are required elsewhere.

(U) The RRTs shall be released from their area of operation on a rotational basis so that
the ones having gone first are also relieved first.

(V) The lodging and boarding arrangements including food shall be made by the district
administration. For this activity the JD/CVOs of the concerned districts shall
establish liaison with the District Magistrate and ensure that necessary steps have
been taken.

 

LIST OF DISEASES AND SAMPLES TO BE COLLECTED

A laboratory diagnostic service may be of assistance to the Veterinarian, as it will enable him to
arrive at a more accurate diagnosis and may provide information of value in instituting therapy or preventive measures. However, the success of laboratory examination depends mainly on the proper collection, preservation and dispatch of adequate and suitable material. The materials required for diagnosis and the methods to be adopted for their collection and preservation depend on several
factors such as the kind of examination required, the disease under investigation, the apparatus
available, the atmospheric conditions and the length of interval between collection and laboratory examination. All possible measures should be taken for specimen to reach the laboratory in the shortest possible time and as nearly as possible, in the same condition as at the time of their
collection. Whereas the field officer can view the entire carcass and note the condition of all the
organs, the laboratory worker will have available only the material supplied to him for examination.

For meaningful results, the samples must have the following qualities:

1. Correct sampling
2. Correct preservation
3. Correct labeling and identification
4. Correct packing

 

General Considerations

 

White collecting material for the laboratory examination, the filed officer should have the following considerations.

1. The specimen from an animal in the advanced stages of the diseases is most desirable. If the
   disease is a flock or herd problem, specimens should be collected from more than one
   diseased or dead animal. In such flocks or herds submit specimens from one or two animals
   that are in various stages of illness.
2. The materials from ailing 5 to 6 or more animals should be collected at the height of body
   temperature / clinical signs.
3. When sero-diagnosed desired always paired sterile, about 2 ml sera should be collected. One
   serum sample at the time of start of disease and another after recovery (3-4 weeks) from
   disease.
4. The specimen submitted should be characteristic of the disease as seen in the field.
5. In collecting specimens every attempt should be made to avoid contamination with intestinal
   contents, hair and dirt etc.
6. Collect tissues in sterile containers, sealed and transported on sufficient ice to the nearest
   laboratory for storage and processing.
7. Small tissue pieces of ½ x 2 cms thick from organs showing the lesion are considered better
   for preservation and fixation in 10% formalin.
8. All specimens  collected  in  bottles  should  be  sealed,  labeled  clearly  indicating  the
   fixative/transport media used.
9. Care should be taken to seal and pack these bottles in hard boxes/polythene bags to avoid
   leakage during transit.

 

 

Identification of the Samples

 

In the covering letter, all particulars and a completer history including the following should be submitted with each sample by the filed officer.

 

1. Owners name and address

 

2. Description of  the  animal/bird  including  species
   tag/brand/tattooing etc.
3. Duration of the condition or outbreak
4. Morbidity rate
5. Mortality rate
6. Clinical signs
7. Clinical diagnosis (Disease suspected)
8. Treatment history
9. Time of animal’s death and that of necropsy
10. Necropsy rreport

,  age,  sex,  colour,  number  of  ear

 

11. Nature of feed including any change of feed that has occurred in recent past
12. Possibility of contact with animals of neighbouring farms
13. Specific tests required
14. Type of preservative used
15. Veterinarian’s name address and telephone number

For quick disposal of the material, it is advisable to forward one copy of the letter by post and to enclose another in the parcel containing the specimen.

 

 

Preservation of Specimens

 

Fresh tissue which is left in a warm environment (at room temperature) will become liquefied with a foul odour, mainly due to autolysis and putrefaction. The examination of fresh specimen therefore requires the action of a preservative to prevent such deterioration. The preservation of cells and tissue constituents in as life like manner as possible is essential. The choice of preservative will be governed by the type of investigation required.

 

Methods of Preservation

 

 

1. Refrigeration:

(i) Natural Ice: This method of refrigeration is adequate only if the samples are properly packed and the distance to the laboratory is not great. Ice will preserve specimens for 18 to 24 hours during the winter months but only for 8 to 12 hours during hot weather. . Specimens packed in ice should be placed in a water container and surrounded by ice, either in the form of frozen cans of water or by packing a small container into a larger one containing ice, which may be refilled at intervals as desired. For the purpose a thermos flask may be used.

(ii) Dry Ice: This method of refrigeration is preferred if the specimen can be frozen without interfering with the laboratory procedures to be conducted. The specimen should be placed in plastic or other waterproof container and the dry ice wrapped in paper and placed in the box. Do not place the dry ice in direct contact with the specimen unless freezing is not a problem.

Do not send dry ice in an air proof metal containeras the ice will volatilize and pressure may result in an explosion.

Note: Materials collected for bacteriological examination should not be kept at sub zero temperature
(-20⁰C) while for virus isolation these can be stored at -20 to -80⁰C. For most of the diseases keep at
4⁰C.

 

2. Chemical Preservatives:

These preservatives save the specimens from decomposition and such specimens should not be used for bacterial examination.

These solutions are used when bacterial growth is to be kept to a minimum:

1. Formalin 10% 2. Alcohol 70% 3. Phenol 0.5%            4. Merthiolate 1: 10000
2. Glycerol Saline 50%may be used for specimens for viral isolations, however, frozen specimens are preferred.

 

Histopathological Examination:

 

Histopathological examination is done on fixed tissues. Fixation is the process of killing and
hardening tissue. The aim of fixation is the preservation of cells and tissue constituents in as life-
like manner as possible and to allow the preparation of tissue to cut thin sections.
No single substance or known combination of substances has the ability to preserve and allow the
demonstration of every tissue component. Because of this, some fixatives have only special and

limited application and most are mixtures of two or more reagents designed to make use of the special features of each. The choice of fixative will depend upon the type of investigation required. One fixative will rarely be suitable for a variety of methods. it is therefore, convenient to divide them in different groups according to their uses.

 

1. Micro-anatomical Fixative: These are used when it is desired to preserve the anatomy of tissues with correct relation of tissue layers and large aggregate of cells to one another. Most of the routine work of pathological histology is done with such fixatives eg. Formal Saline, Buffered Formal Saline, Alcoholic Formalin, Buffered Gluteraldehyde, Zenkers Fluid, Bouin’s Fluid, Susa’s Fluid Gendre’s Fluid, Rossman’s Fluid.
2. Cytological Fixatives: These are used when the preservation of intracellular structures or
   inclusions is of first importance. Often these elements are preserved at the expense of even
   penetration, ease of cutting and loss of other cell structures. Cytological fixatives are of two types:

(i) Nuclear Fixatives: Carnoy’s Fluid, Clarke’s Fluid, Flemming’s Fluid New Comer’s Fluid

(ii)  Cytoplasmic  Fixative:  Champy’s  Fluid,  Muller”s  Fluid,  Helly’s  Fluid,  Regaud’s  Fluid, Schaudinn’s Fluid, Formal Saline and Formal Calcium.

 

3. Histochemical Fixative: When histochemical tests are to be applied, it is essential that the fixative employed produces minimum changes in the element that is to be demonstrated. Freeze drying technique is ideal for this purpose, but it is very troublesome and time consuming for routine work. eg. Formal Saline, Cold Acetone, Absolute Alcohol.

The fixative of choice for specimens for histopathological examination is 10% Formal Saline. This preservative is prepared by diluting formaldehyde (40% stock solution) 100 ml (1 part) to 900 ml (9 parts) of normal saline solution (NSS 8.5%). For fixation of tissues, a sufficiently large quantity of formalin should be used, approximately 10 times as much preservatives as tissue.

 

Virological Examination:

 

Most of the known viruses affecting animals have a tendency for selective tissue localization and thus demand the utmost care in selecting specimens appropriate for the disease and as free as possible from bacterial contamination.

 

The transport media used specially for virological examination of the morbid materials are 50% phosphate buffered saline (pH 7.3-7.4) and Hank’s balanced salt solution. 50% buffered glycerine saline is the general preservative for animal inoculation tests. For other tests especially for tissue culture, tissue samples should be fresh, refrigerated but not frozen. Blood sample sent without chilling should be collected in EDTA and 50% glycerine saline. Swabs for viral isolation should be placed into tissue culture fluid or isolation media containing antibiotics.

 

Tissue specimens may be placed in sterile, wide mouth, cork-stoppered bottles and forwarded frozen in dry ice or in 5 to 10 volumes of sterile 50% glycerol saline or preferably in a medium containing equal parts of pure buffered glycerine. The material should reach the laboratory in the shortest possible time after collection, preferably over ice in a thermos flask. When a viral disease is suspected antibiotics (Penicillin 1000 units and streptomycin 10 mg/ml) should be used in transport media and serum samples dispatched for diagnosis. This will inhibit contamination. About 20 gm each of spleen/lymph node tissues be collected for virus isolation. Always 5 to 6 or more animals be investigated and material collected for laboratory examination.

 

Scabs from pox may be forwarded in a dry sterile specimen tube without addition of a preservative. Materials from animals suspected for Rinderpest, PPR or Canine Distemper should not be collected in glycerol saline. It should be sent in sterile vials with ice.

Heart blood, serum and cerebrospinal fluid for identification of viruses should be forwarded in refrigerated sterile vials.

In case suspected for rabies in dog or other small animals, the entire head should be submitted in a water tight metal container placed in a larger container refrigerated with natural ice or dry ice. If the distance to the laboratory is too great for the specimen to arrive in a refrigerated condition, the brain should be removed and divided between the cerebral hemispheres; one hemisphere should be placed in undiluted neutral glycerine and the other in 10% formalin.

 

The small dead bird should be immersed in a 5% lysol solution, wrapped in lysol soaked cheese cloth and forwarded frozen in dry ice.

 

 

Type of Materials to be Collected in Different Viral Diseases:

 

Foot and Mouth Disease: Samples of epithelial debris from fresh lesions or the aspirated contents of unruptured vesicles is 50% buffered sterile glycerine saline preferably on ice. About 10 ml blood at the hight of body temperature in EDTA/Heparin. Tonsils and in calves pieces of heart in 10% formalin and ice separately.

 

Rinderpest/Bovine virus Diarrhoea : (1) From live animals, about 10 ml or more blood at the
height of body temperature in anticoagulant, rectal swab in PBS on ice, nasal and pharygeal
secretions, faeces (2) From dead animals, prescapular lymph nodes, spleen, (20-30 gm) on ice and

(3) Lung, liver, spleen, tonsil etc. in 10% formalin. Materials 20 gm from 5 to 6 or more animals be collected and dispatched for better picture of disease/outbreak.

Peste des Petits Ruminitas (PPR): Pieces of intestine on ice.

 

Mucosal Disease: Nasal swabs, tissues or blood samples refrigerated immediately after collection.

 

Swine Fever: (1) Heparinised 20 ml blood in sterile vials or test tube on ice from live animal (2)
Heart blood, pieces of spleen lymph node, pancreas (10 to 15 gm) in 50% buffered glycerine saline

(3) Pieces of brain, lung, intestines,  Ileocaecal region and kidney in 10% formalin from dead
animal. Material from 5 to 6 or more animals be collected in order to give diagnosis/ true picture of
disease. Materials for isolation and serological tests may be collected in sterile vial on ice without
glycerine.

Transmissible Gastroenteritis (Swine): Faeces, jejunum-ileum.

 

Malignant Catarrhal Fever: Lymph nodes and spleen preferably in ice or in 50% glycerine-saline,
blood in O.C.G. or heparin or sodium citrate and all tissues in 10% formalin for histopathology.

Blue Tongue: Blood taken in Sodium Citrate or EDTA, lesion material, spleen and regional lymph
nodes.

 

Rabies: Whole intact head in ice or half portion of brain in 50% glycerine saline in water tight hard box and the rest half portion of brain in 10% formalin as well as salivary glands . Alternative and preferable, small pieces from hippocampus and brain (cerebellum, medulla, cerebrum, spinal cord) in 50% buffered glycerine and 10% formalin separately duly sealed in bottles and packaged in thick polybags and hard box, labeled “Suspected for Rabies” Where available fresh smears from brain may be stained with Seller’s stain.

Pseudorabies: Brain, lesion material, regional lymph nodes.

 

Pox (Sheep, Goat, Cow & Buffalo): Scab in sterile container on ice, scab in 50% buffered glycerine, skin lesions in 10% formalin, separately.

 

Bovine Herpes Virus 1,2,3/IBR/IPV, Bovine Mammilitis/Parainfluenza 3/Adenovirus. etc>:

Paired serum (sterile) samples on ice (IBR/IPV, Bovine Mammilitis) (2) Swabs from vaginal and nasal lesions and pieces of trachea, lung in transport medium on ice (3) Smears amd pieces of trachea, liver, turbinate bone, lung in Bouin’s fixative/10% formalin.

African Horse Sickness/Arbo Viruses: Blood at the height of temperature, in heparin (5-10 units/ml) or EDTA, paired sera samples in sterile container on ice. About 10 ml blood and 2 ml serum add antibiotic or merthiolate may be collected on ice. Dead animals- (1) Spleen, lymph nodes, intestine, internal organs etc. in 10% formalin.

Caprine Artheritis/Encepahalitis/Maedi/Visna Disease: Paired sera samples, joint capsule, lung, brain on ice and 10% formalin.

Canine Distemper: Pieces of lung, urinary bladder, liver, trachea, stomach, wall and brain in 10% formal saline. Impression smears from liver, piece of liver and spleen on ice.

Influenza (Equine, Porcine and Avian): Nasal swab in PBS or Hanks medium on ice, paired serum, lung, tracheal mucosa, nasal pharyngeal secretion.

 

Equine Infectious Anaemia (EIA): Paired sera samples, all internal organs in 10% formalin.

Equine Arteritis Virus Infection: Whole blood, nasal and pharyngeal secretions, placenta and foetus, regional lymph nodes, spleen.

 

Infectious Canine Hepatitis: Liver, gall bladder and kidney in 10% formalin-saline. Impression smears from liver fixed in methanol. Spleen and liver in sterile containers on ice.

 

Canine Parvovirus: Rectal swab in PBS, pieces of intestine, heart on ice, all internal organs in 10% formalin.

Rift Valley Fever: Whole blood.

 

Ranikhet Disease: (1) Freshly dead/moribund bird on ice (2) Portion of liver, spleen, trachea, bronchi, lung in 50% buffered glycerine saline on ice (3) Proventriculus in 10% formal saline.

Marek’s Disease: (1) Live bird in acute stage of disease (2) Feather follicles from chest and neck in transport medium (3) Paired sera samples (4) Portion of peripheral nerve, trachea, ovary, liver, kidney, spleen and skin in 10% fromalin for histopathology.

 

Infectious Bursal Disease (Gumboro Disease): (1) Live affected chick/bird (2) Bursa of fabricious in transport medium (3) Paired sera samples (4) Bursa of Fabricious, kidney, spleen in 10% formalin for histopathology.

 

Infectious Bronchitis/Other Respiratory Diseases: (1) Swab from exudate, lung (2) Paired sera
samples.

 

For diagnosis of poultry diseases it is desired that a few ailing/moribund/dead birds may be sent for collection of suitable material at the laboratory)

 

Bacteriological Examination:

 

The material must be collected with aseptic precautions and dispatched in sterile containers to prevent contamination from extraneous sources. In case of dead animals, the specimens shluld be taken soon after seath to avoid the chances of invasion of tissues by putrefactive bacteria rendering the specimens unsuitable for examination.

Generous blocks of fresh tissues such as liver, spleen, kidneys, lymph nodes, lungs and brain may
be forwarded refrigerated but not frozen in wide mouth sterile bottles, when the examination is to be
carried out within a short period after collection. If, however, the examination is to be delayed by a
few days, it is preferable to preserve the tissues in 25% glycerine saline. While forwarding material
in this preservative, it would be better to send larger pieces of the tissues, so that if necessary,
cultures may be attempted from their central areas where the glycerine penetration has been the
least.

 

Liquid material such as heart blood, cerebrospinal fluid and inflammatory exudates may be taken either on sterile swabs, sealed in pipettes or collected in tubes or bottles with strict aseptic precautions. The surface of the organ should be well seared with a hot spatula and a sterile syringe and needle or pipette inserted into it for drawing the material. If the peritoneal fluid is required, an area on the abdominal wall is seared thoroughly and a sterile knife is used for holding the cavity open and sterile pipettes for drawing the fluid.

For taking swabs of the contents of a closed abscess in a living animal, clip the hair from the area
and paint it with tincture of iodine. Open the abscess with a sterile scalpel and take swabs from the
wall as well as from the contents of the abscess and keep the swabs in sterile tubes. To collect
wound discharge, the wound should be thoroughly cleaned with warm water and soap, and sterile
non-antiseptic cotton wool or gauze dressing applied. The material should be collected after about

24 hours by inserting a sterile swab underneath the dressing.

 

For bacteriological examination of the intestinal flora, ligate about 6 inches of the bowel at both ends and forward the loop unopened under refrigeration.

If there are abnormal contents in the uterus, a small segment is isolated between ligatures and retained unopened for bacteriological examination. While it is desirable that the specimen for immunological study be refrigerated in transportation, serum for agglutination and complement fixation test may be preserved with 0.5% phenol or 1:10000 merthiolate.

 

 

Type of Materials to be Collected in Different Bacterial Diseases:

 

Anthrax: Cotton swabs soaked in exuded blood/blood taken from a superficial ear vein, in acute and per acute anthrax.

In swine, organisms are not present in blood, so swabs should be taken from exudates and the cut surface of hemorrhagic lymph nodes.

Flame fixed blood smears of cattle and sheep. From subcutaneous swelling in horses, swine and dogs. Swabs of blood from ear vein for cultural examination from dead animals. A small piece from tip of ear or muzzle (1/2 x 1 cm approx.) in saline or without any preserve in sterile glass test tube or bottle on ice duly sealed. It is not advisable to  open the carcass suspected for anthrax in the field. If opened, it should be properly disposed by burning.

Blackleg (Black Quater): Impression smears from the affected muscle tissue; exudate from lesions; pieces of affected muscles on ice.

Tetanus: Material from wound site (isolation is not usually attempted) Bacillary Haemoglobinuria: Affected liver tissue.

Botulism: Suspected food, meat, forage and urine.

Enterotoxaemia, Lamb Dysentery: Intestinal pieces with contents inside tied with thread or contents of small intestine with and without chloroform separately on ice, kidney, urine.

Listeriosis: (1) Aborted foetus, brain, placenta (2) All internal organs in 10% formalin/on ice (3) Half brain under refrigeration and half in 10% formal saline

Vibriosis (pigs): Affected portion of intestine.

 

Campylobacteriosis: Prepeutial washings, semen, foetal stomach contents, cervicovaginal mucus (sample should reach the laboratory within 6 hours of collection under refrigeration or at room temperature in transport media).

 

Brucellosis: Paired sera samples, blood and abomasal contents of aborted foetus, placenta with 2-3 cotyledons, vaginal swabs in PBS, separate bottle on ice, whole foetus if small on ice. For ABR milk is used avoiding colustrum and milk from drying off animals or those suffering from mastitis. Milk, serum, vaginal mucous etc. from dam.

 

Haemorrhagic Septicemia: From sick animals fixed smears from blood and throat swelling and from dead animals, smears from heart blood and liver. Heart blood in a sterile pipette/bottle, lymph node and spleen on ice.

Johne’s Disease: Rectal pinch smears, bowl washings (at least 10 gm preserved in 10% formalin).
In dead animals terminal portion of ileum with iliocaecal valve, mesentric lymph gland in 10%
formal-saline.

Glanders: Exudate from skin and ung lesions in vials on ice. Impression smears from exudate duly fixed, tissue containing early nodules, pus from ulcers.

 

Tuberculosis: (1) Cough material in sterile container from live animal (2) Sample of milk in sterile
container (3) Suspected lesions in 10% formal saline (dead animal) (4) Smears from lesions fixed
by heat (5) Lymph glands or lung lesions in sterile container for isolation in 50% buffered
glycerine.

 

Leptospirosis: (!) Blood serum (2) Pieces of liver and kidney in 10%  formalin (in dead animals) and (3) Milk or urine in vials by adding 1 drop of formalin per 20 ml.

Salmonella Sp.: Intestinal swab, heart blood, bile in sterile container on ice.

 

Actinomycosis & Actinobacillosis: (1) Smears from pus lesions, pus in vial on ice (2) Formalin preserved materials from lesions (affected muscle)

Mycoplasmosis/CCPP/CBPP/Coryza: (1) Swab from lesions, nasal and vaginal swabs in PBS on ice (2) Piece of lung preserved in 10% formalin for histopathological examination and on ice separately. Paired sera samples.

 

Chlamydia/Psittacosis: (1) Nasal swab, lung pieces in sterile container on ice and internal organs
in 10% formalin (2) Fixed impression smears from liver, lung and foetus (3) Paired sera samples

 

Mycotic Infections: Deep skin scrap in sterile vials

 

Skin Diseases (Ring Worm, Mange, Mites): Skin scrappings for identification of ectoparasites and fungus in vials.

 

 

Parasitological Specimen:

 

For all parasitological specimens 5-10% formalin of formal saline and 70% alcohol are used as
preservatives. After collection, ectoparasites and intermediate hosts can be sent as such or after
fixation. 70% alcohol does not retain the colour of ticks as well as does the chloroform formalin
mixture. The latter is prepared by adding chloroform  in excess to 10% formalin and shaking it
thoroughly. The chloroform is allowed to settle and the top solution is poured constituting the
chloroform formalin mixture which retains the natural colour of ticks, if dropped alive.

Fleas and lice are collected on a sheet of paper either by combing or with the help of a camel hair brush moistened with xylene. These insect forms are best preserved in 70% alcohol, with 5% formalin as a second choice. Sufficient exudates or scabs and crusts formed from exudate in 70% alcohol and deep skin scrappings (until petechial hemorrhages appear) in 5% formalin are collected if mites are suspected. Skin pieces are collected in 10% formalin.

 

The collection of fleas, lice and mites from skins of small animals and birds is best accomplished by
brushing, combing and shaking the fur, hair or feathers over a large sheet of white paper and placing
them in a vial containing 70% alcohol. Dead small birds and animals can be transported as such in
securely tied polythene bags. The ectoparasites can also be collected by dipping and washing the
birds in a pail with water containing small amount of detergent. The parasites are collected with the
help of a strainer. Larval forms should be collected and placed in a vial of 70% alcohol or 5%
formalin.

Common blood protozoan parasites can be visualized in thin blood smears. For demonstration of
microfilariae thick blood smears, whole blood or hemolysed (with acetic acid) centrifuged blood is
desirable.

 

The  trophozoite  forms  of  intestinal  protozoa  seldom  survive  in  dead  animal  and  at  room
temperature. Faeces or intestinal contents as such or in normal saline must, therefore, be examined
immediately after collection or retained at body temperature till examined. Protozoan cysts are
comparatively resistant and can be identified in faeces for 3-4 days. Refrigeration further preserves
them.

The best all round preservative for helminths is 5-10% formalin or formal saline. Samples of faeces
should be examined for the presence of helminth ova as soon as possible after collection or put in
hot 10% formalin (one part formalin: 3 part faeces) to prevent development and hatching of eggs
and decomposition of sample. The sample of faeces to be cultured must be free from earthy or
bedding contamination otherwise it may be heavily infected with free living nematodes ot their
larvae.

Alive worms are washed by shaking in normal saline and then put for about an hour in 70% alcohol, normal saline or water at 37⁰ to 40⁰C to cause them to die in an extended state. Dead worms are fixed in 5% formalin and may later be transferred to 70% alcohol. The final preserving fluid, either 70% alcohol or 5% formalin, should contain 5% glycerine to prevent drying of helminth worms through evaporation of preserving fluid.

 

Theileriosis: (1) Biopsy smears from swollen lymph nodes from early stages of disease fixed with
methanol. Blood smears fixed in methanol or alcohol. Two to three blood smears from each case.

Babesiosis: Thin blood smears from early stages of disease fixed with methanol. Two to three blood smears from each case.

Anaplasmosis: Thin blood smears from ear vein fixed with methanol. Two to three blood smears from each case.

Surra/Trypanosomiasis: (1) Blood in anticoagulant on ice (2) Blood smears fixed in methanol.

 

Gastro-Intestinal Parasitic Disease: (1) Faecal sample in 10% formalin and (2) in dead animals,
parasites (round worms in 70% formalin) for identification. All internal organs in 10% formalin.

 

Serological Samples:

 

 

Paired samples (one taken at the onset of the disease and another taken 2-3 weeks later) aree desirable. For anaplasmosis it should be frozen or preserved with 0.2% Beta propiolactone. In other cases, it may be preserved with sodium merthiolate 1:10000 or carbolic acid 1:200.

Immunoflourescense Samples: Serum as such and freshly collected, refrigerated or frozen tissue. Biopsy Samples:

1. Bone marrow smears for hemopoietic disorders.
2. Lymph nodes for lymphosarcoma (whole or part)
3. Liver biopsies for histopathology or chemical estimation
4. Skin biopsy in 10 times volume of 10% formal saline
5. Skin scrappings for mite infestation and hair samples for fungal infections are sent without any preservatives or in 5% formalin.

Blood Samples:

1. Blood smears for hemopoietic disorders or blood infections shluld be fixed in methyl
   alcohol, dried and sent.
2. Blood samples for haematology-Anticoagulants viz. EDTA 2mg/ml, Sodium Citrate 3.8% –
   1ml/10ml, Hellar and Paul formula – (0.8 g Potassium oxalate, 1.2 Ammonium oxalate,
   water 100cc), Heparin – 0.1 IU/ml or 1 mg/5 ml (not good for rabbit blood)
   For prothrombin time estimation Sodium citrate or Potassium oxalate is used.

 

Anticoagulants for Blood:

 

1. Heparin 5-6 i.u./ml of blood
2. Oxalate phenol glycerine (OCG) solution-1 part to 2 parts of blood erythrocytes.
3. Ethylene Diamine Tetra-acetic (EDTA) at the rate of 1-2 mg/ml of blood.

 

Blood is transported in chilled condition, but never frozen for both clinical examination as well as virus isolation. Where bacterial isolation is not required, antibiotics may be added.

 

Cerebrospinal Fluid:

Examination soon after collection is desirable. For leucocytes EDTA and for glucose estimation sodium fluoride is added as a preservative.

Synovial Fluid: One clotted (2-3 ml) and one unclotted sample (2 ml) with EDTA or Sodium citrate be submitted.

Serous Cavity Fluid: No preservative is required and used for bacteriological or cytological examination.

Sputum: No preservative is required and is used for bacteriological or parasitological examination.

 

Toxicological Examination:

 

In case suspected for poisoning, each specimen should be forwarded for laboratory examination in a
separate wide mouth glass stoppeered bottle or a jar under refrigeration without the addition of a
preservative. The material submitted to a laboratory should include stomach with its contents after
tying both ends; about 30 cm each of ileum and colon and their contents with their ends tied. In the
case of ruminants, about 1 kg of well mixed contents of rumen; about 0.5 kg of liver; one or both
the kidneys and adipose tissue, contents of urinary bladder, blood or other specified material and
specimens of plants suspected for poisoning and also specimens that have been dried in shade.

 

In cases that may result in Vetero-legal action, particular care should be exercised for safe possesion from the time the specimens are collected until they are delivered to the toxicologist. The type of poison suspected should be stated to assist in the laboratory diagnosis. All bottles and packings should be carefully sealed by the officer making the examination, closed in such a manner that they cannot be opened without destroying the seal.

When an officer forwards articles to the chemical examiner or toxicologist for examination, he should at the same time address and forward separately a letter to the chemical examiner regarding their dispatch, the letter should contain:

1. An impression of the seal used in closing
2. A list of articles forwarded and information as to how the articles have been forwarded.
3. The name of the Officer from whom the order has been received to forward the articles, and
   the number and date of such order.
4. Information as to the number and kind of animals affected and the number of deaths.
5. Any information obtained on post mortem examination, nature and duration of symptoms
   which may be likely to indicate the probable nature of the poison.

 

Aflatoxicosis: (1) Suspected feed (specially groundnut cake) about 100 g each (2) Piece of liver (50g), spleen in 105 formal saline and on ice separately

 

Poisoning Cases: (1) Stomach and intestinal contents 100 g on ice (2) Left over fodder 100 g

(3) About 100 g liver pieces in alcohol on ice.

Forage Poisoning: Samples of grass/fodder, plants, liver and stomach contents on ice.
Poisoning:

 

S. No.	Suspected Poison	Required Material (In order of importance)
1.	Arsenic (Acute)	1. Liver 2. Kidneys 3. Stomach Contents
2.	Arsenic (Chronic)	1. Hair 2. Liver 3. Urine
3.	Alkaloids	1. Liver 2. Urine 3. Brain 4. Stomach Contents
4.	Copper	1. Liver
5.	Cyanide	1. Stomach Contents 2. Liver 3. Oxalated Blood
6.	Insecticides (Chlorinated)	1. Fat 2. Liver 3. Stomach Contents 4. Lymphoid Organs
7.	Insecticides (Organophosphate)	1. Oxalated Blood 2. Liver 3. Stomach Contents 4. Lymphoid Organs
8.	Lead (Acute)	1. Kidneys 2. Liver 3. Urine
9.	Lead (Chronic)	1. Hair 2. Liver 3. Kidneys 4. Urine
10.	Mercury	1. Liver 2. Kidneys 3. Stomach Contents 4. Intestinal Contents
11.	Nitrate and Nitrite	1. Stomach Contents 2. Whole Blood
12.	Phosphorous	1. Stomach Contents 2. Whole Blood 3. Oxalated Blood
13.	Phenols-cresols	1. Liver 2. Stomach Contents 3. Kidneys
14.	Rodenticides	1. Stomach Contents 2. Liver 3. Urine
15.	Strychnine	1. Stomach Contents 2. Urine 3. Liver 4. Brain
16.	Sodium Chloride	1. Oxalated Whole Blood 2. Brain 3. Stomach Contents

The  following  are  the  minimum  quantities  of  the  specimens  to  be  sent  for  toxicological

examination:

 

 

S. No.	Name	Amount
1.	Blood	30 to 50 ml
2.	Brain	Entire
3.	Fat	200 g
4.	Hair	5-10 g
5.	Intestinal Contents	one
6.	Kidneys	500 – 1000 g
7.	Liver	500 – 1000 g
8.	Stomach Contents	500 – 1000 g in large animals and all available contents in small animals
9.	Urine	All available

 

Miscellaneous Condition:

 

1. Mastitis: Milk samples in sterile tubes on ice.
2. Abortion: Whole foetus on ice or all internal organs, vaginal swab in PBS or Hanks, pieces
   of palcents in sterile containers on ice and in 10% formalin separately. Paired sera samples.
3. Infertility and Sterility: Sterile semen, prepucial swab and paired sera sample on ice.
4. Pyrexia: Blood smears, blood in EDTA and paired blood serum on ice.

Note:

1. In general for diseases of unknown etiology it is essential to collect feed, blood smears and
   blood serum from live animals. In dead animals stomach contents, spleen lungs, liver,
   kidney, intestine in sterile containers and 10% formalin separately. Specimen packed for
   rabies , glanders and anthrax be marked “suspected for”
2. About 10 ml blood, 6 ml sterile serum, 20 g tissues be collected for virus isolation. Examine

5 – 6 animals for collection of materials.

3. Paired Serum: One serum (2 ml)at the time of onset of disease and another 3 weeks after
   first collection when animals almost recover from disease.
4. The Veterinary  staff  attending  the  dogs/suspected  cases  of  animal  rabies  should  be
   immunized with anti-rabies vaccine.
5. Use washing soda and soap for washing of floor.
6. All instruments be kept in boiling water for 30 minutes after post mortem examination.
7. All requests for investigation should be sent by the Officer incharge preferably through
   Director/Additional Director/Joint Director etc. along with full details of disease suspected,
   its gravity and the officer to be contacted for further correspondence/or for intimation of
   results.

 

 

Washing and Sterilization:

 

 

(1) Glasswares: All glasswares used for collection of material are first washed with warm water.
Used glasswares are first autoclaved for 30 minutes at 15 pounds pressure (presusure cooker). Wash
with detergent or 3% washing soda at 80⁰C. Use soft brush for removing protein. Vim or soap may
be used on outer surface of glassware only. After washing in running water 10 times, the glassware
may be washed in glass distilled or metal distilled water 3 times. Dried in air or oven. Tubes and
bottles may be plugged with cotton cloth. Aluminium foil and paper be used for packing of
glassware. All the glasswares are sterilized at 180⁰C for one hour in hot air oven/Autoclave/Pressure
cooker.

 

(2) Transport Media: Sterilize at 15 lbs for 30 minutes in autoclave or pressure cooker and store at
4⁰C.

 

Preparation of Fixatives/Media?Preservatives: Formal Saline Solution

37-40% Formalin                                            –           100 ml

Sodium Chloride                                             –               9 g

Tap Water                                                       –           900 ml

 

Buffered Neutral Formalin Solution

37-40% Formalin                                            –           100 ml

Distilled Water                                                –           900 ml

Sodium Phosphate Monobasic                        –               4 g

Sodium Phosphate Dibasic (anhydrous)         –            6.5 g

 

Formalin Sodium Acetate Solution

37-40% Formalin                                            –           100 ml

Sodium Citrate                                                –             20 g

Tap Water                                                       –           900 ml

Formalin Ammonium Bromide Solution

 

37-40% Formalin, neutralized                         –            15 ml

Ammonium Bromide                                      –              2 ml

Distilled Water                                                –            85 ml

Formalin Alcohol Acetic Acid Solution

37-40% Formalin                                            –              5 ml

Alcohol 80%                                                   –            90 ml

Glacial Acetic Acid                                        –              5 ml

 

Formol Calcium Solution

Calcium Chloride Anhydrous                         –             1 ml

37-40% Formalin                                            –           10 ml

Distilled Water                                                –           90 ml

Zenker’s Solution                                          –

Distilled Water                                                –       1000 ml

Mercuric Chloride                                           –           50 ml

Potassium Dichromate                                    –           25 ml

Sodium Sulphate                                             –           10 ml

 

Bouin’s Solution

Picric Acid (Saturated Aqueous Solution)     –           50 ml

37-40% Formalin                                            –         250 ml

Glacial Acetic Acid                                        –           50 ml

 

Formal Sublimate

Formalin                                                          –         100 ml

Saturated Solution of Mercuric Chloride       –         900 ml

Carnoy’s Solution

Absolute Alcohol                                            –           75 ml

Glacial Acetic Acid                                        –           25 ml

 

Clarke’s Solution

Absolute Alcohol                                            –           75 ml

Glacial Acetic Acid                                        –           25 ml

 

Newcomer’s Solution

Isopropanol                                                     –           60 ml

Propionic Acid                                                –           30 ml

Petroleum Ether                                              –           10 ml

Acetone                                                           –           10 ml

Dioxane                                                           –           10 ml

 

Orth’s Solution

Potassium Dichromate                                    –           2.5 ml

Sodium Sulphate                                             –              1 ml

Distilled Water                                                –           100 ml

Mix and Add: Formalin 37-40%                    –             10 ml

Hank’s Balanced Salt Solution (HBSS)

HBSS Dry Powder – 1 vial (Micro lab or Himedia or any make)

Distilled Water – 1000 ml

Dissolve the powder and add 0.5 g Gelatin powder. Sterilize at 15 lbs pressure for 30 minutes. Cooll

and  add  sterile  sodium  bicarbonate  to  make  pH     7.4  and  antibiotics   (Penicillin   1000  iu/ml

Streptomycin 10 mg/ml). Store in 3 ml quantity in vials at 4⁰C for collection of swabsand fluid for

virus isolation.

Merthiolate and Sodium Azide Solution:

 

0.001% concentration of either of the two is good preservative for serum used for serological test.
But not for serum used for neutralization test. Use antibiotics when serum neutralization test is
desired.

 

M/25 Phosphate Buffer Glycerol (pH 7.6):

Solution A: M/25 Disodium Hydrogen Phosphate: A quantity of Na₂HPO 12 H₂O is spread out and
dried in an incubator for 3 or 4 days. 7.13 g of the dried salt is made up of 1 liter with double
distilled water.

Solution B: M/25 Potassium Dihydrogen Phosphate; 5.45 g of KH₂PO₄ is made upto 1 liter with double distilled water.

Buffer Solution: 6 parts of solution A+1 part of solution B gives abuffer solution of approximately
pH 7.6.

Mix the buffer solution with equal volume of pure glycerol and adjust it to pH 7.6.

 

Phosphate Buffer Saline (PBS), pH 7.2

Sodium Chloride                                             _            8.5 g

Di-sodium Hydrogen Phosphate                    –           0.56 g

Potassium Dihydrogen Phosphate                  –           0.14 g

 

Nutrient Broth (Ready made dehydrated media are also available. Follow instructions given

on bottle):

Beef Extract (Lab-lemco)                               –           10 g

Peptone                                                           –           10 g

Sodium Chloride (NaCl)                                 –             5 g

Distilled Water                                                –     1000 ml

Oxalate Phenol Glycerine (OCG) Solution:

 

Potassium Oxalate                                          –       500 ml

Glycerine                                                         –       500 ml

Carbolic Acid                                                  –           5 g

Distilled Water                                                –       500 ml

Stains:

Ready made BDH/E, Merk Giemsa/Gram’s/Ziehl Nelson Acid fast stains are locally available.

 

 

 

Decalcification:

Perenyi’s Fluid

 

10% Nitric Acid Aqueous                              –           40 ml

Absolute Alcohol                                           –           30 ml

0.5% Chronic Acid Aqueous                          –           30 ml

Nitric Acid Method

1. Place calcified specimen in large quantities of nitric acid solution until decalcification is complete (Change solution daily for best results).

 

 

Instruments/Other Items Needed for Collection pf Specimens:

 

(a) Surgical Instruments:

Scissors 6″ long – 2 Nos., Forceps 6″ – 2 Nos., Sharp Scalpel or Blade Holders with BP Knife – 2; these should be sterilized in autoclave or oven. Each instrument should be packed separately.

 

(b) Gloves:

Latex gloves of different sizes.

 

(c) Pasteur Pipettes:

Rubber bulbs of 30 ml capacity to be fitted in pasteure pipettes for collection of body fluids.

 

(d) Bottles/Vials:

5 ml clear screw capped McCartney/Biju bottles with rubber stoppers aand metal screw cap. Presterilized disposable bottles may be used. Empty bottles/vials after washing and sterilization may be used. Sterile non breakable bottles are also available in the market.

 

(e) Microscopic Glass Slides:

Standard size being 7.7 cm x 2.5 cm and 1.1 mm thick with smooth margins.

 

(f) Swabs:

Cotton swabs in strong bamboo sticks (15 cm long) are prepared and sterilized in individual tubes for collection and transport of body fluids.

(g) Syringes:

Glass/disposable plastic syringes of 5/10 ml size are used for collection of blood and body fluids.
For large animals 16 SWG needles and for small animals 20 SWG 30 mm long needles should be
used.

(h) Sealing and Labelling Tape Rolls

This should be used for sealing the vials containing serum/blood/other fluids etc. and for labelling the materials. The details of materilas should be written by water proof ink.

 

(i) Packing Boxes:

Light, strong wooden boxes of approprate size for keeping the bottles/vials.

(j) Sterilized Absorbent/Non Absorbent Cotton:

Absorbent  cotton  for  making  the  swabs  and  non  absorbent  cotton  for  plugging  the  test
tubes/bottles/vials and also to be used as packing material for protecting the glasswares during
transportation.

(k) Polythene Bags:

Thick polythene bags of assorted size for keeping the stomach contents/morbid tissues/faeces etc. and transported on ice.

(l) Protective Garments:

Aprons, gum boots and face mask.

 

INFORMATION TO BE PACKED ALONG WITH THE MATERIAL

 

 

1. Owners Name & Address :
2. Description of the Source Animal :
3. Nature of Container Used : Sterile/Unsterile
4. Preservative / Transport Medium Used :
5. Anticoagulants Used in Blood :
6. Disease Suspected :
7. Clinical Signs / History :
8. Necropsy Findings (In Brief) :

 

MATERIALS BEING SENT

1. Blood : Heart Blood/Peripheral Blood
2. Smear : Blood/Pus/Impression Smears
3. Swabs : Nasal/Eye/Rectal/Vaginal/Others
4. Serum : Single/Paired
5. Scrappings : Skin/Pock Lesions/Others
6. Excretions : Faeces/Vomitus/Urine/Others
7. Discharges : Eye/Nasal/Mouth/Rectal/Vaginal/Others
8. Epithelium : Tongue/Lips/Gums/Foot/Others
9. Others :

 

LABORATORY EXAMINATIONS DESIRED

 

10. Bacteriological Examination
11. Virological Examination
12. Parasitological Examination
13. Toxicological Examination
14. Histopathological Examination

: Heart Blood/Discharges/Tissues/Any Others : Heart Blood/Tissues/Skin Scabs/Others

: Blood/Blood Smear/Faeces/Others
: Liver/Stomach Contents/Feed/Fodder/Others : Name the Tissues Being Sent

 

(i)______________ (ii) ______________ (iii) _______________ (iv) ______________

(v) _____________ (vi) _____________ (vii) ______________ (viii) ______________

 

 

Signature of the Officer In-charge

Name:

Designation:
Seal

Format of letter to be sent along with clinical/morbid material for laboratory diagnosis

Letter No.:_______________________________    Dated: _______________________________

 

From:

__________________________________
__________________________________
__________________________________
__________________________________
__________________________________

To,

The Coordinator

Animal Disease Diagnostic Center

 

Through:________________________________________________________________________

I am sending herewith the material for laboratory diagnosis. The details of the materials are given
below:

1. Owner’s Name & Address: ________________________________________________________
2. Description of the source of animal: _________________________________________________ Species __________, Breed __________, Age/Date of Birth ________________, Sex _________
   Identification No. _____________
3. Date & Time of Death: ___________________________________________________________
4. Date & Time of Collection of Material: ______________________________________________

 

5. Brief History of the Case:

Duration of Illness/Outbreak:

Clinical Signs/Symptoms Observed: Morbidity/Mortality Rate:

Treatment History:

 

 

Necropsy Findings (In Brief):

 

6. Nature & Contents of Specimen (Type of Material):

Blood/Serum/Faeces/Urine/Skin Scrapping/CSF/Synovial Fluid/Biopsy: Morbid Material for Histopathology:

(1) ________________   (2) _________________  (3) _________________

(4) ________________   (5) _________________  (6) _________________

Plant/Feed Material:
Other Material:

7. Preservative/Transport Media Used:
8. Examination(s) Desired:
9. Any Other Information/Remark

Signature:____________________

Name:_______________________

Seal:

 

DISINFECTION PROTOCOL TO BE FOLLOWED AT THE TIME OF EVACUATION OF PERSONNEL INVOLVED IN RELIEF WORK IN NON ZOONOTIC DISEASE OUTBREAKS

 

 

A disinfection center is to be set up at a place located at the periphery of the area in which the outbreak is occurring under the supervision of an officer of the department.

The center shall have several rooms and bathrooms with facilities of hot water.

All personnel engaged in the treatment of affected animals shall mandatorily report to this center while moving out of the affected area.

They shall be provided with large PVC garbage bags to pack their soiled/dirty clothing which shall be sealed with packing tape or rubber bands.

Shoes shall also be cleaned thoroughly with available disinfectant with a brush and then air dried. If possible a separate pair of shoes/slippers may be used while coming out of the area and the old pair packed and sealed in the PVC bag.
Thereafter a freshly washed pair of clothes shall be put on.

After reaching their respective place of posting all personnel shall not engage in any clinical work for a period of 24
hours.

Vehicles used for relief work when moving out of the area shall be thoroughly cleaned with 1% phenyl applied for a period of 20-30 minutes before being rinsed with water.

An officer shall be deputed to man this center and shall ensure that every body is following the laid down protocol.

QUARANTINE AND DISINFECTION PROTOCOL TO BE FOLLOWED AT THE TIME OF EVACUATION OF PERSONNEL INVOLVED IN RELIEF WORK IN ZOONOTIC DISEASE OUTBREAKS

 

The following protocol shall be strictly enforced and followed:

As and when replacements arrive the personnel who have completed 21 days first shall be replaced first to the quarantine center and shall remain there for a period of one week and/or the reported incubation period of the disease. The Veterinary Officers serving in and posted in the districts having the outbreak will not be relieved from their posts. Since them being more familiar with the territory shall help in guiding and implementing relief operations. This shall continue till such time that the outbreak is bought under control.

In addition to the above the Veterinary Officers posted in non clinical areas like (Chief Technical Officers, Veterinary Officers in laboratories, Veterinary Officers in Offices, etc.) shall also be mobilized for replacement
A disinfection and quarantine center is to be set up at a suitable place immediately in the periphery of the affected area under the supervision of an officer of the department.

The center shall have several rooms and bathrooms with facilities of hot water.

All personnel engaged in active treatment of animals shall mandatorily report to this center while moving out of the
affected area. All officer and staff inclusive of Veterinary Officers, Livestock Extension Officers, Pharmacists,
Dressers, Attendants, technicians, drivers, porters, support staff and vehicles shall have to undergo rigorous disinfection
before evacuation from these areas. This is very important from the point of accidental spillage of the disease to other
areas.

They shall be provided with large PVC garbage bags to pack their soiled/dirty clothing which shall be sealed with packing tape or rubber bands. Concomitantly if facilities exist then the clothes can be boiled/autoclaved and air dried and then packed in large PVC bags and sealed with tape.

Shoes shall also be cleaned thoroughly with available disinfectant and or 2% acetic acid with a brush and then air dried.
If possible a separate pair of shoes/slippers may be used while coming out of the area and the old pair packed and sealed
in the PVC bag.

An application of 0.2% citric acid on the whole body surface followed by a hot water bath with a strong carbolic soap. Thereafter a freshly washed pair of clothes shall be put on.

Thereafter they will remain in quarantine for a period of seven days at this center or for the number of days of the incubation period for the disease in question.

After reaching their respective place of posting all personnel shall not engage in any clinical work for a period of 24
hours.

Vehicles used for relief work when moving out of the area shall be thoroughly cleaned with 1% phenyl applied for a period of 20-30 minutes before being rinsed with water.

An officer shall be deputed to manage this center and shall ensure that all personnel follow the laid down protocol.

 

List of Scheduled Animal Diseases

(a) Multiple Species Diseases
1. Anthrax
2. Aujeszky’s disease
3. Bluetongue
4. Brucellosis
5. Crimean Congo haemorrhagic fever
6. Echinococcosis/hydatidosis
7. Foot and mouth disease
8. Heartwater
9. Japanese encephalitis
10. Leptospirosis
11. New world screwworm (Cochliomyia hominivorax)
12. Old world screwworm (Chrysomya bezziana)
13. Paratuberculosis
14. Q fever
15. Rabies
16. Rift Valley fever
17. Rinderpest
18. Trichinellosis
19. Tularemia
20. Vesicular stomatitis
21. West Nile fever
(b) Cattle Diseases
1. Bovine anaplasmosis
2. Bovine babesiosis
3. Bovine genital campylobacteriosis
4. Bovine spongioform encephalopathy
5. Bovine tuberculosis
6. Bovine viral diarrhoea
7. Contagious bovine pleuropneumonia
8. Enzootic bovine leucosis
9. Haemorrhagic septicaemia
10. Infectious bovine rhinotracheitis/Infectious pustular vulvovaginitis
11. Lumpy skin disease
12. Malignant catarrhal fever
13. Theleriosis
14. Trichomoniasis
15. Trypnosomiaisis
(c) Sheep and Goat Diseases
1. Caprine arthritis/encephalitis
2. Contagious agalactia
3. Contagious caprine pleuropneumonia
4. Enzootic abortion of ewes (ovine chlamydiosis)
5. Maedi-visna
6. Nairobi sheep disease
7. Ovine epididimitis (Brucella ovis)
8. Pestes des pestes ruminitas
9. Salmonellosis (S. abortusovi)
10. Scrapie
11. Sheep pox and goat pox
(d) Equine Diseases
1. African horse sickness
2. Contagious equine metritis
3. Dourine
4. Equine encephalomyelitis (Eastern)
5. Equine encephalomyelitis (Western)
6. Equine infectious anaemia
7. Equine influenza
8. Equine piroplasmosis
9. Equine rhinopneumonitis
10. Equine viral arteritis
11. Glanders
12. Surra (Trypanosoma evansi)
13. Venezuelan equine encephalitis
(e) Swine Diseases
1. African swine fever
2. Classical swine fever
3. Nipah virus encephalitis
4. Porcine cysticercosis
5. Porcine reproductive and respiratory syndrome
6. Swine vesicular disease
7. Transmissible gastroenteritis
(f) Avian Diseases
1. Avian chlamydiosis
2. Avian infectious bronchitis
3. Avian infectious laryngotracheitis
4. Avian mycoplasmosis (M. gallisepticum)
5. Avian mycoplasmosis (M. synoviae)
6. Duck virus hepatitis
7. Fowl cholera
8. Fowl Typhoid
9. Highly pathogenic avian influenza and low pathogenic avian influenza in poultry
10. Infectious bursal disease (Gumboro disease)
11. Marek’s disease
12. New Castle Disease
13. Pullorum disease
14. Turkey rhinotracheitis
(g) Lagomorh Diseases
1. Myxomatosis
2. Rabbit haemorrhagic disease
(h) Other Diseases
Camelpox
Leishmaniosis