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In general the following points should be duly considered while collecting materials for laboratory diagnosis of bacterial, viral, parasitic and other diseases.

a. All materials collected should be accompanied with full history of disease out break viz., morbidity and mortality rates, clinical signs, duration of disease, species affected, disease suspected etc.

b. The materials from ailing 5-6 or more animals should be collected at the height of body temperature/ clinical signs.

c. When sero-diagnosis is 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 (3-4weeks) from disease.

d. All biological specimens should be transported on ice to the nearest laboratory though a courier within shortest possible time where they may be processed and stored on proper conditions. The courier should be instructed to change ice if long distance is involved.

e. When death is recorded in ailing animal, the post-mortem examination should be conducted at the earliest to avoid putrification of the carcasses. Putrified materials are unfit for lab examination.

f. Materials collected for bacteriological examination should not be kept at subzero temperature (-20 ̊ C) while for virus isolation these can be stored at -20 to -80̊ C. For most of the diseases keep at 4̊ C.

g. Detailed post-mortem report should be despatched with morbid materials in 10% formalin. The morbid materials should be sent without preservative in sterile containers over ice. The transport media used specially for virological examination of the morbid materials are 50% Phosphate Buffered Glycerine, Phosphate Buffer Saline (pH 7.3-7.4) PBS and Hank’s Balanced Salt Solution. In case of non-availability of transport media, it is always desirable to collect tissues in sterile containers, sealed and transported on sufficient ice to the nearest laboratory for storage and processing. Small tissue pieces of ½x2 cms thick from organs showing the lesions are considered better for preservation and fixation in 10% Formalin. The specimen bottles should be sealed, labelled clearly indicating the fixative/transport media used. Care should be taken to seal and pack these bottles in hard boxes/polythene bags to avoid leakage during transit. Unbreakable bottles and sterile polybags may be used for collection and transport of biological specimens. When viral disease is suspected, antibiotics (Penicillin 1000 units & streptomycin 10mg/ml) may be used in transport media and in serum samples despatches for diagnosis. This will inhibit contamination. About 20 gram each of spleen/lymphnode tissues be collected for virus isolation. Always 5 to 6 or more animals be investigated and material collected for lab examine. Less number of materials sometimes fail to give results/true picture of disease outbreak. The type of materials to be collected in different diseases is mentioned below:

BACTERIAL DISEASE

1. HAEMORRHAGIC SEPTICAEMIA: From sick animals fixed smears from blood and liver. Heart blood in a sterile pipette/bottle, lymph node and spleen on ice.

2. ANTHRAX: 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 muzzle (½x1 cm approx.) in saline or without any preservative in sterile glass test tube or bottle on ice duly sealed. It is not advisable to open the carcass suspected for anthrax in field. If opened, it should be properly disposed by burning.

3. BLACK QUARTER : Impression smears from the affected muscle tissue; exudate from lesions; pieces of affected muscles on ice.

4. ENTEROTOXAEMIA,LAMB DYSENTERY : Contents of small intestine with and without chloroform separately on ice, kidney, urine.

5. BRUCELLOSIS : Paired serum, 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.

6. JOHNE’S DISEASE : Rectal pinch smears, bowl washings(atleast 10gm preserved in 10% formalin). In dead animals terminal portion of ileum with ileocaecal valve, mensenteric lymph gland in 10% formal-saline.

7.GLANDERS : Exudate from skin and lung lesions in vials on ice. Impression smears from exudate duly fixed.

8. 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 and (5) Lymph glands or lung lesions in sterile container for isolation in 50%buffered glycerine.

9. LEPTOSPIROSIS : (1) 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 20ml.

10. SALMONELLA SP. : Intestinal swab, heart blood, bile in sterile container on ice.

11. ACTINOMYCOSIS & ACTINOBACILLOSIS : (1) Smears from pus lesions, pus in vial on ice (2) Formalin preserved materials from lesions (affected muscle).

12. LISTERIOSIS: (1) Aborted foetus, brain, placenta (2) All internal organs in 10% formalin/on ice.

13. 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 serum.

14. 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.

15. MYCOTIC INFECTIONS: Deep skin scrap in sterile vials.

16. SKIN DISEASES (RINGWORM, MANAGE, MITES : Skin scrapings for identification of ectoparasites and fungus in vials.

VIRAL DISEASES

1. RINDERPEST/RINDERPEST ALLIED DISEASE/PPR/BOVINE VIRUS DIARRHOEA: (1) From live animals: About 10ml or more blood at the height of body temp, in anticoagulant, rectal swab in PBS on ice. (2) From dead animals: Prescapular lymph nodes, spleen, (20-30g) on ice and (3) lung, liver, spleen, tonsil etc. 10% formalin. Materials 20g from 5 to 6 or more animals be collected and despatched for better picture of disease/ outbreak. Eye, mouth and rectal swab in PBS on ice. Pieces of intestine on ice for PPR.

2. FOOT AND MOUTH DISEASE: Vesicular fluid from unruptured oral vesicles and curetted epithelium from fresh lesions oesopharyngeal fluid in 50% Phosphate Buffered Glycerine preferably on ice. About 10ml blood at height of body temp. In EDTA/Heparin, Heart pieces on ice. At least 5 or more animals be investigated and material collected. Pieces of heart in calves 10% formalin and ice separately.

3. RABIES: Half portion of brain; salivary gland in 50% Phosphate Buffered Glycerine or Glycerine Saline in water tight hard box and the rest halt portion of brain in 10% formalin. Alternatively small pieces from Hippocampus and Brain (Cerebellum, medulla cerebrum, spinal cord) in 50% Buffered Glyerine and 10% formalin separately duly sealed in bottles and packed in thick polybags and hard box Labelled “Suspected for Rabies:”. Where available fresh smears from brain may be stained with Seller’s stain.

4. POX (SHEEP, COW AND BUFFALO): Scab in sterile container on ice, scab in 50% Buffered Glycerine. Skin lesions in 10% formalin, separately.

5. BOVINE HERPES VIRUS 1,2,3/IBR/IPV, BOVINE MAMMILTIS/ PARAINFLUENZA 3/ ADENO VIRUS ETC. Paired serum (sterile) on ice (IBR/IPV,Bovine Manilitis), Swabs from vaginal and nasal lesions and pieces of trachea, lung in transport medium on ice., and Smears and pieces of trachea, liver, turbinate bone, lung in Bouin’s fixative/10% formalin.

6. SWINE FEVER: Heparinised 20ml blood in sterile vials or test tube on ice form live animal, Heart blood, pieces of spleen, lymph node, pancreas (10 to 15g each) in 50% Buffered Glyerine Saline, pieces of brain, lung, intestines, ileocaeacal 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 glyerine.

7. BLUETONGUE DISEASE/AFRICAN HORSE SICKNESS/ARBO VERUSES: Blood at the height of body temperature, in heparin (5-10 units/ml) or EDTA, paired sera in sterile container on ice. About 10ml blood and 2ml may be collected on ice. Dead animals-Spleen, lymph nodes (5-6g) on ice Spleen, lymph nodes, intestine, internal etc. 10% formalin.

8. CAPRINE ARTHRITIS/ENCEPHALITIS/MAEDI/VISNA DISEASE: Paired serum, joint capsule, lung, brain on ice and 10% formalin.

9. CANINE DISTEMPER: Pieces of lung, urinary bladder, liver, trachea, stomach wall and brain in 10% formal-Saline, Impression smears from liver, Piece of liver & spleen in ice.

10. EQUINE INFLUENZA Nasal swab in PBS or Hanks on ice, paired serum.

11. EQUINE INFECTIOUS ANAEMIA (E.I.A): Paired serum, all internal organs in 10% formalin.

12. INFECTIOUS CANINE HEPATITIS: Liver, gall bladder and kidney in 10% formalin-Salina. Impression smears from liver fixed in methanol. Spleen and liver in sterile containers on ice.

13. CANINE PARVOVIRUS: Rectal swab in PBS, pieces of intestines, heart on ice, all internal organs in 10% formalin.

14. RANIKHET DISEASE: Freshly dead/moribund bird on ice. Portion of liver, spleen, trachea, bronchi, lung in 50% Buffered Glycerine cerol-Saline in ice and Proventriculus in 10% Formal-Saline.

15. MAREK’S DISEASE: Live bird in acute stage of disease Feather follicles from chest and neck in transport medium Paired serum and Portion of peripheral nerve, trachea, ovary, liver, kidney, spleen and skin in 10% formalin for histopathology.

16. INFECTIOUS BURSAL DISEASE (GUMBORO DISEASE): Live affected chick/bird, Bursa of fabricious, kidney, spleen in 10% formalin for histopathology.

17. INFECTIOUS BRONCHITIS/OTHER RESPIRATORY DISEASES: Swab from exudates, lung Paired sera. (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)

PARASITIC DISEASES

 
1. THEILERIOSIS: Biopsy smears from swollen lymph nodes from early stage of disease fixed with Method. Blood smears fixed in Methanol or alcohol. 2-3 blood smears from each case.

2. BABESIOSIS: Thin blood smears from early stage of disease fixed with Methanol 2-3 blood smears.

3. ANAPLASMOSIS: Thin blood smears from ear vein fixed with methanol. 2-3 blood smears.

4. SURRA/TRYPANOSOMIASIS: Blood in anticoagulant on ice, Blood smears fixed.

5. GASTRO-INTESTINAL PARASITIC DISEASES: Faecal sample in 10% formalin IN dead animals, parasites (round worms in 70% formalin) for identification. All internal organs in 10% formalin

TOXICOSES/ POISONINGS


1. AFLATOXICOSIS: Suspected feed (specially groundnut cake) about 100gm. Each, Piece of liver (50g), spleen in 10% Forma saline and on ice separately.

2. POISONING CASES: Stomach and intestinal contents 100gms. On ice, Left over fodder 100gm and about 100gm liver pieces in alcohol on ice.

3. FORAGE POISONINGS: Sample of grass/fodder, plants, liver and stomach contents on ice.

MISCELLANEOUS CONDITIONS

1. MASTITIS: Milk samples in sterile tubes on ice.

2. ABORTIONS: Whole foetus on ice or all internal organ, vaginal swab in PBS or Hanks, pieces of placenta in sterile containers on ice and in 10% formalin separately. Paired serum.

3. INFERTILITY AND STERILITY: Sterile semen, prepucial swab and paired serum in 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, blood and serum from live animals. In dead animals stomach contents, spleen, lung, lymphnode, liver, kidney, intestine in sterile containers and 10% formalin separately. Specimens packed for Rabies, Glanders and Anthrax be marked “suspected for”

2. About 10 ml blood, 6 ml sterile serum, 20 gm tissues collected for virus isolation. Examine 5 to 6 animals for collection of materials.

3. Paired serum: One sera (2ml) 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 immunised with Anti-rabies vaccine.

5. Use washing soda and soap for washing f 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 through Director/Joint Director/Deputy Directors etc. Along with full details of disease suspected its gravity and the officer to be contacted for further correspondence /for intimation of results.