Sample collection methods in different veterinary diseases

COLLECTION PRESERVATION AND DISPATCH OF VETERINARY SPECIMEN FOR LABORATORY DIAGNOSIS

Tissue samples are collected from dead or live animal for laboratory examination to confirm the tentative diagnosis.

PURPOSE:   

1. Diagnosis of the disease.
2. Confirmation of tentative diagnosis
3. Prognosis
4. To observe the effect of treatment and give direction for future therapy.

Sample collection in veterinary diseases

GENERAL CONSIDERATIONS  FOR COLLECTION  OF SPECIMENS: 

1. The diseases most commonly encountered in animals are of bacterial, viral, parasitic, fungal and metabolic origin. Diagnosis based on symptoms and laboratory examination of the relevant materials is essential for initiating treatment at the proper time. In general the following points should be duly considered while collecting materials for laboratory diagnosis.
2. All materials collected should be accompanied by full history of disease outbreak namely species affected, duration of disease, clinical signs, morbidity and mortality rates, disease suspected etc.
3. The collected biological specimens should be transported on ice to the nearest laboratory as early as possible.
4. Materials collected for bacteriological examination should be kept at refrigeration temperature (4 °C) in case of delay of transportation. If a viral etiology is suspected the material can be stored at –20°C to -80 °C.
5. When sero-diagnosis is required, collect paired serum samples (about 2 ml sera). One serum sample should be collected at the onset of disease and second sera after recovery (3-4 weeks) from disease preferably on 21st day.
6. If death is reported, the post-mortem examination should be conducted at the earliest as putrefied materials are unfit for laboratory examination & detailed post-mortem report should be attached along with the samples collected during post-mortem.
7. For Histopathological studies, tissues should be preserved in 10% formalin. The volume of formalin used should be approximately 10 times the volume of material. Specimen bottles with wide mouth should be used for collecting tissues.
8. All the impression smears before packing, should be fixed in methanol for 1-5 minutes unless otherwise specified.
9. In case of outbreaks, try to collect materials from as many ailing animals (5-6 or more) as possible at the height of body temperature /clinical signs.

Collection Procedure of different biological material /specimen

 

Name of specimen	Collection Procedure
Blood	Blood: Blood is examined for different purposes as listed below :   ·     Preparation of blood smears for microscopic examination:                                         The ear vein is the most convenient site for taking blood when required for the preparation of smears or films. In poultry, blood is taken from comb, wattles or wing veins. ·     For cultural examination, transmission experiments and other studies: In cows, buffaloes, horses, sheep and goats, blood is preferably taken from the jugular vein. From pig, blood is usually obtained by cutting the end of the tail or tip of the ear, or from anterior vena cava. In dogs and cats, from the saphenous or radial vein and from fowls brachial vein (on the ventral surface of the wing).From ducks jugular vein is the preferred site. The site should be prepared by removing hair /feathers and disinfect it with spirit. Blood is collected by sterile syringe. From post-mortem cases, blood is collected from right ventricle. For serological studies, serum is separated by allowing the blood to clot, keeping the syringe in a slanting position. After complete separation, serum is transferred to a sterile vial. For haematological studies, blood is not allowed to clot by using anticoagulant (sodium citrate or EDTA). One part of anti-coagulant is added to 9 parts of blood.
Milk:	Milk for bacteriological examinations should be collected in sterile container. For isolation purpose; milk should be collected after discarding the first few streams of milk.
Faeces	Sample for examination of parasitic eggs or coccidial oocysts are collected from the rectum or at the time of defecation.
Urine:	Urine is collected at the time of micturition or obtained with the help of a clean and sterile catheter.
Pus:	Smear is made on a clean glass slide. For cultural examination, it should be obtained directly from the abscess in a sterile container, or sterile swab is used.
Nasal/throat/uterine  discharges:	Samples should be collected on sterile cotton swabs, then placed in sterile screw cap test tubes.
Exudates/transudates and body fluids	Collection has to be made with the help of sterile pipettes or syringes and to be despatched in leak proof sterile screw cap containers.
Tissue, blood and exudates smears:	Tissue smears are to be prepared from cut surfaces of organs and fixed over a flame. Blood/exudates smears are drawn as thin film and fixed over a flame or in methyl alcohol.

 

Preservation of materials for specific examination

Specific examination	Preservatives used
Bacteriological/ Mycological	·     Blood and exudates: are to be collected aseptically with sterile Pasteur pipettes/syringes, and then put in sterile tubes or vials without any preservative and to be transported over ice. Especially for Anthrax and HS blood should be collected in sterile container /Cryo vials with (500-1000µl EDTA is sufficient) and without EDTA. After collection cotton soaked with alcohol should be placed on the collection site and burned and transported strictly under cold chain. ·     Tissues: Pieces of affected organs with lesions should be collected in sterile condition and should be transported under cold chain.
Virological	·     Blood and exudates: to be collected aseptically without any chemical preservative and to be stored and transported on ice. In some viral infections (e.g. Blue Tongue and Classical Swine Fever blood may be collected in vials/vacutainer containing EDTA). ·     Tissues: Pieces of affected organs are to be collected under aseptic condition and transported either on ice. For FMD 50% buffered glycerine saline is the preferred transport medium
Parasitological	·     For identification of parasite and helminth ova 10% formalin is the preferred transport medium to preserve the integrity of the ova.. ·     For coccidial oocysts, 2.5% Potassium dichromate solution is the preferred transport medium.
Serological tests:	·     After collection, serum samples have to be stored at -20°C and should be transported strictly under cold chain.
Histopathological	·     For routine and general histopathological examination tissue pieces are to be collected in 10% formal saline (collection to be done in wide mouth bottles with10 times the volume of tissues) ·     A copy of detailed post-mortem report should be sent.
Toxicological                                ( Forensic laboratory)	·     For chemical analysis fresh tissues and fluids should be sent as soon as possible and on ice. Avoid addition of preservatives to the samples. Use 95% ethanol @ 1ml per gram of sample when necessary.

 

SPECIMENS REQUIRED FOR LABORATORY EXAMINATIONS IN FEW IMPORTANT DISEASES

Bacterial diseases
Name of Disease	Antemortem sample	Postmortem sample
Anthrax:	Smear from ear vein, discharges or from swelling, Whole blood with and without EDTA.	Smear of blood from the ear vein or caudal vein Whole blood  should  be collected from ear vein/caudal vein Exudate or blood mixed soil should be placed in sealed pack Pieces of ear, or muzzle should be placed in sterile container for Ascoli’s test.
Haemorrhagic Septicaemia	Smear from ear vein Smear from fluid of swelling of throat. Whole blood from jugular vein with or without EDTA	Blood smears or exudates obtained with the help of sterile syringe and fluid from oedematous swelling. Heart blood with and without EDTA Portion of Lungs, spleen and mediastinal lymph nodes, liver on ice Long bones. Lungs & other affected tissue in 10% formalin
Black quarter  /Black leg	Smear from affected muscle or swab from the swelling of the affected quarter by making small incision Whole blood from jugular vein with or without EDTA	Smear from swelling Affected muscle piece on ice.
Actinomycosis /  Actinobacilosis	Pus from mandible and maxilla & Pus smear. Slides from pus for sulfer granules.	Tissue from affected parts in 10% formalin Pus in sterile test tube/from edge of lesion. Portion of swelling mass on ice
Brucellosis	Serum after 3 weeks of abortion. Vaginal mucus; seminal plasma; uterine discharge	Foetal stomach tied off Pieces of stomach, liver, placenta, aborted foetus on ice
Mastitis	10 ml milk in sterile vial on ice	Blood smear, milk and pus from the affected quarter, if available
Tuberculosis	Sputum in a sterile swab, sample of milk from infected udder Faeces in a sterile container	Smear from lymph glands showing nodular lesions Lungs, lymph glands, intestine, mesenteric lymph glands should be collected if lesions are seen on ice
John’s Disease	Rectal pinch, swab or smear	Bowel washing in sterile bottle Smear from rectal mucosa Mesenteric lymph node in 10% formalin Smear from thickened areas of intestine, small portion of bowel showing gross lesions
Glanders	Nasal discharge; pus from skin lesions	Smear from discharge Lung, liver and spleen in 10% formalin Serum
Strangles	Smears from pus of abscess; smears from discharge from nasal cavity	Smear from pus of affected part. Pieces of affected lymph nodes, lungs, spleen, and trachea on ice
Salmonellosis	Blood is collected from wing vein Faeces in sterile container	Intestinal content from ileum and large intestine on ice Mesenteric lymph nodes, kidney and gall bladder on ice
Leptospirosis	Serum 21 days after abortion. Blood ( at febrile stage ) on ice Milk / urine in Vials( 1 drop of formalin in 20 ml sample	Portion of kidney & liver on ice
Listeriosis	CSF and whole blood with and without EDTA	Half brain in ice. Half brain in 10 % formalin
Colibasilosis	Faeces in sterile container	Abdominal viscera; heart blood, fresh intestinal tissue in Sterile vial
Tetanus	Smear from the exudates/wound	Muscles, spinal cord and brain on ice
Enterotoxaemia	Whole blood with or without EDTA	Small intestinal contents, intestinal smear
Mycoplasmosis / CCPP/CBPP	Swabs from sinus/ trachea Nasal and vaginal swabs preferably in Amies transport medium on ice Serum samples (paired serum)	Piece of lung preserved in 10% formalin and on ice separately
Rabies	Saliva on ice if possible	Brain on ice for demonstration of viral antigen, viral inclusions and isolation of virus.
F.M.D	Vesicular fluid Tongue epithelium from areas of ruptured vesicles in sterile vials containing 50% buffered glycerine Oesophageal and pharyngeal fluid, collected by probang with mouth gag.	.Lymph nodes, kidney, adrenal gland, heart &Thyroid gland on ice
PPR	Ocular, buccal, rectal, nasal swabs on ice, no preservative should be added. Paired serum samples	Pieces of spleen, lymph nodes, lungs, liver on ice. Lungs, liver, spleen, tonsil in 10% formalin for HP
Pox	.Pox scab on ice	Pieces of skin and other organ with lesion in 10% formal saline
Classical Swine Fever	Whole blood in EDTA on ice is the most preferred sample	Pieces of spleen, mesenteric lymph glands, intestine especially ileocaecal region in 50% glycerol saline for isolation. Pieces of brain, lung, intestines, ileocaecal region & kidney for HP
Ehrlichiosis	Blood smear, whole blood in EDTA	Spleen and lungs on ice
Ring worm	Skin scrapings from the lesions, including some hair roots, unpreserved in a tight container.	Skin scrapings from the lesions, including some hair roots, unpreserved in a tight container
Mange	Scabs and deep skin scrapings	Portion of skin showing lesion
Poisoning Cases	Blood samples on ice Stool, vomitus & urine sample on ice.	Stomach and intestinal contents should be sent after proper ligation at both the end sent it in ice to avoid putrefaction Liver spleen, kidneys should be sent in separate container on ice. Feed sample should be sent in separate container on ice

Dispatch of Samples to Animal Health Laboratories:

Following points must be kept in mind while dispatching the material to laboratory for diagnosis:

• Describe the clinical signs, lesions, tentative diagnosis and treatment given to animal in your letter; also mention the type of test you want with your tentative diagnosis.
• Write correct address on letter as well as on the parcel preferably with pin code, if the material is sent through post.
• Mark the parcel “Biological Material” “Handel with Care” “Glass Material” etc .in order to avoid damage in parcel, also mark the side to be kept on upper side with arrows.
• Seal the container so that it should not leak in transit.
• A separate specimen box should preferably be used for each case so that they do not contaminate each other, or leak in transit..
• Try to send the material as soon as after collection from animal.
• Keep one copy of cover letter inside the parcel and another copy by hand or post in a separate  cover
• Put a frozen coolant pad into each tin when packing the specimens for despatch.
• In poisoning cases the address of Forensic Laboratory should be clearly written.
• The bottle or container should be sealed and labelled properly indicating the name of owner, identification of animal (tag no /mark/colour/sex/ breed /age/species) type of tissue collected and preservative used (if any).The examination requested and disease or poisoning suspected should also be written.
• A copy with details of post-mortem report and containing above information should be sent separately under separate cover
• All the container should be packed with cloth and sealed with sealing wax and should be sent through a person in order to avoid any breakage in transit.