flash

Login Forum

Status

Tarikh Kemaskini : 10/04/2014

 

UNTUK KEGUNAAN KAKITANGAN SAHAJA

FIELD GUIDE TO SUBMISSION OF SPECIMEN

[ General Notes ]  [ Laboratory tests ] [ Pathology ] [ Avian Submissions ] [ Parasitology & Hematology ] [ Bacteriology] [ Selection of Tissues For Microbiology ] [ Submission of Samples For Serological Tests ] [ Virology ] [ Serology/Virology ] [ Toxicology ] [ Rabies Submission ]
[ Submission of Specimens According to Main Clinical Signs ]


INTRODUCTION

The role of a diagnostic laboratory is to complement the work of the field officer; to assist him in arriving at a diagnosis or to confirm a tentative diagnosis made in the field so that appropriate action can be taken to prevent losses and thus increase productivity. Certain general principles and recommendations should be followed if specimens submitted are to be of any value for laboratory investigation.

VISION OF DEPARTMENT OF VETERINARY SERVICES

The animal industry becomes a fully integrated commercial sector producing quality food from animal products towards national self-sufficiency and export.

MISSION OF DEPARTMENT OF VETERINARY SERVICES

We are committed to the growth of a sound animal industry to supply quality and safe food from animal products for national consumption and export through the provision of quality veterinary services from motivated, skilled and creative individuals.

OBJECTIVES OF DEPARTMENT OF VETERINARY SERVICES

1. To prevent, control and eradicate animal and zoonotic diseases
2. To promote the growth and development of a sound animal industry.
3. To ensure that food of animal origin are clean, wholesome and fit for human
    consumption
4. To promote the growth and development of animal feed industry
5. To ensure the welfare and wellbeing of all animals.

MISSION OF REGIONAL VETERINARY LABORATORY SERVICES

The mission of the Regional Veterinary Laboratory (RVL) is to promote healthy livestock and companion animals and to ensure safe animal products for the consumer by assisting farmers, veterinarians, their clients, and others responsible for animal health in the detection and prevention of disease by providing accessible, accountable, timely and accurate diagnostic services.

FUNCTIONS OF THE LABORATORIES

1. Diagnosis of animal diseases
2. Monitoring of animal health status
3. Monitoring of pathogens
4. Provide disease information
5. Provide support for training to entrepreneurs in animal industry and
    departmental staffs in animal health.

PURPOSE OF THIS MANUAL

The purpose of this manual is to guide veterinary practitioners and assistants in selections, preservation, and delivery of specimens to the RVL for more timely and accurate evaluations and tests. We hope that this guide will be useful to you in your daily routine of attending to animals in good health, but much more importantly when diseased or deceased. On our part we can only assess its value by an improvement in the quality of future submissions and by your response in the form of criticisms which will help us to improve this guide.

LOCATIONS OF REGIONAL VETERINARY LABORATORIES IN MALAYSIA

The laboratory services are available in six strategic locations in Peninsular Malaysia. The locations of the RVLs are as follows:

(1) RVL Bukit Tengah - serving the northern states of the Peninsular Malaysia such as Perlis, Kedah, Penang and north Perak.
(2) RVL Petaling Jaya - serving the central states of the Peninsular Malaysia such as south Perak, Selangor, Negeri Sembilan, Malacca, east Pahang and Kuala Lumpur.
(3) RVL Johor Baharu - serving the southern states of the Peninsular Malaysia such as Johore.
(4) RVL Kuantan - serving the east coast states of the Peninsular Malaysia such as Pahang, Kelantan and south Terengganu.
(5) RVL Ipoh (attached to the Veterinary Research Institute) - serving the state of Perak
(6) RVL Kota Bharu - serving the east coast states of the Peninsular Malaysia such as Kelantan and north Terengganu.

In addition to the six RVLs in Peninsular Malaysia, there are also one Animal Disease Research Centre (ADRC) in Sabah and one State Veterinary Diagnostic Laboratory (SVDL) in Sarawak. RVL Kota Bharu is a reference laboratory for FMD monitoring and diagnosis using ELISA to detect antigen and antibody. Veterinary Research Institute (VRI) also acts as a reference laboratory for RVL for further laboratory test and confirmation. Certain tests such as Brucella CFT, Johne's CFT, Leptospira MAT, Nipah virus ELISA and Salmonella serotyping are carried out in VRI.

DIRECTORY OF VETERINARY LABORATORIES

Makmal Veterinar Kawasan Bukit Tengah,
P.O.Box 63, 14007 Bukit Mertajam.
Penang.
Tel:  04-5072540
Fax: 04-5075796
Email: 
Makmal Veterinar Kawasan Petaling Jaya,
Persiaran Barat,
46630 Petaling Jaya. Selangor.
Tel:  03-7572963
Fax: 03-7574421
Email: 
   
Makmal Veterinar Kawasan Johor Bahru,
P.O.Box 734, 80730 Johor Bahru.
Johore.
Tel:  07-2239243
Fax: 07-2242528
Email: 
Makmal Veterinar Kawasan Kuantan,
Jalan Sri Kemunting 2,
25100 Kuantan. Pahang.
Tel: 09-5137400
Fax: 09-5134949
Email: 
   
Makmal Veterinar Kawasan Kota Bharu,
16150 Kubang Kerian,
Kota Bharu. Kelantan.
Tel:  09-7653754
Fax: 09-7654339
Email: 
Veterinary Research Institute,
59, Jalan Sultan Azlan Shah,
P.O.Box 369, 30740 Ipoh. Perak.
Tel:  05-5457166
Fax: 05-5463368
Email: 
   
Animal Disease Research Centre,
P.O.Box 59, 89457 Tanjung Aru.
Sabah.
Tel:  088-261263
Fax: 088-232488
Email: mikelee@adrcdvs.gov.my
 
State Veterinary Diagnostic Laboratory,
Kota Samarahan,
93632 Kuching. Sarawak.
Tel:  082-611607
Fax: 082613460
 
GENERAL NOTES ON SUBMISSIONS AND EXAMINATIONS, PRESERVATION, PACKING AND DISPATCH OF SPECIMENS
 
 

It is important that all specimens collected should arrive at the laboratory in a state as similar as possible to that in which it existed in the animal body just before death. All forms of distortion should be prevented and include such things as bacterial contamination, autolytic changes and putrefaction.

The specimen submitted should always be correctly labeled. The labeling should not come off in the presence of water or some other liquids.

Packing of the specimens must be done in a manner to prevent leakage so that dissemination of infective agent does not occur. Plastic bags, wax paper and glass container may be used. These should then be suitably packed in boxes/thermos flasks to prevent breakage while in transit. Non-disposable containers will be returned; so make sure that your own address is on them.

The specimens should be sent by the fastest means of transports to the laboratory nearest to you. Specimens should preferably arrive during office hours. Where specimens are due to arrive outside office hours or on public holidays, the laboratory concerned should be notified in advance so that arrangement can be made to collect them.

The appropriate submission form, duly filled in must accompany all specimen submission.

1.  MAKVET 01 Mammalian submission form
2.  MAKVET 02 Avian submission form
3.  MAKVET 03 Serological submission form
4.  MAKVET 04 Feed samples submission form
5.  MAKVET 05 Inter laboratory submission form
6.  MAKVET 05 Rabies sample submission form
7.  MAKVET 06 Submission form for toxicology
8.  MAKVET 07 Laboratory report for all samples
9.  MAKVET 08 Laboratory report for biochemistry
10. MAKVET 09 Submission form for National Pullorum Programme samples
11. MAKVET 10 Aquatic animal submission form
 

 
LABORATORY TESTS FOR EQUINE DISEASES
DISEASES PRESCRIBED TESTS SAMPLES LABORATORY
Equine infectious anemia AGID Sera VRI only
Equine influenza (Virus type A) VI Lung VRI only
Equine rhinopneumonitis SN, VI Sera, Lung VRI only
Japanese encephalitis VI, Haemaglutination inhibition (HI), Dot blot Sera VRI only
Equine herpes virus SN Sera VRI only
Western equine encephalitis SN Sera VRI only
Eastern equine encephalitis SN Sera VRI only
Glanders Agent id. Lymph nodes VRI & all other RVLs
Mallein test Live animal
CF Sera VRI only
Strangles Agent id. Lymph nodes & organs VRI & all other RVLs
Piroplasmosis Agent id. Heparinized blood VRI & all other RVLs
IFA CF Sera VRI only
Surra Agent id. Heparinized blood VRI & all other RVLs
ELISA Sera VRI only
   
  LABORATORY TESTS FOR BOVINE DISEASES
 
 
  LABORATORY TESTS FOR OVINE/CAPRINE DISEASES
 
   
  LABORATORY TESTS FOR POULTRY DISEASES
 
   
  LABORATORY TESTS FOR SWINE DISEASES
 
   
  LABORATORY TESTS FOR AQUATIC ANIMAL DISEASES
   
 
   
  LABORATORY TESTS FOR DISEASES OF MULTIPLE SPECIES
 
  Abbreviation

 

Agent id.

Agent identification
AGID Agar gel immunodiffusion
CF Complement fixation
ELISA Enzyme-linked immunosorbent assay
FAVN Fluorescent antibody virus neutralization
FA Fluorescent antibody
FEC Fecal egg count
HI Haemagglutination inhibition
IFA Indirect fluorescent antibody
MAT Microscopic agglutination test
MRT Milk ring test
NPLA Neutralization peroxidase-linked assay
PCR Polymerase chain reaction
RBPT Rose Bengal Plate test
SN Serum neutralization
VI Virus isolation
SAT Serum agglutination test
 
PATHOLOGY
 
 

CARCASS SUBMISSION

1. General Information

Whenever possible, submit the entire carcass to the laboratory. Ideally, the carcass should be submitted to the laboratory as soon as possible after death. If submission is unavoidably delayed, the carcass should be kept cool but not frozen. Please, DO NOT FREEZE CARCASSES FOR POST-MORTEM EXAMINATION. Freezing produces severe artifacts, which make interpretation difficult or impossible. Please include a complete clinical history with all submissions. It will be most helpful if the attending veterinarian writes the history.

   
  2. Herd Health Problems

In cases involving a herd problem, the best specimen for submission is a live, acutely affected and untreated animal. When submission of live animal is not possible, the second best submission would be samples obtained by the submitting veterinarian at the time of field necropsy. Prior to euthanasia, collect blood in plain (red top) and EDTA (lavender top) tubes. Perform a complete post-mortem examination and submit specimens for microbiology and histopathology as indicated. Submit these samples and a complete history to the laboratory. It is important that the veterinarian selects which animals to submit when there is a herd problem, since the farmer often selects animals, which are not representative of the problem.
   
  TISSUE SUBMISSION
 
1. DO NOT FREEZE SPECIMENS FOR HISTOPATHOLOGY

Freezing produces artifacts in tissues, which generally result in the sections being unsuitable for interpretation. Do not place formalized samples in direct contact with ice bags or frozen specimens as this may cause freezing of the fixed tissue.
   
  2. Fixation: 10% phosphate buffered formalin fixative of choice

RECIPE:
Commercial formaldehyde (37-40%) 100 mls
Distilled water 900 mls
Sodium phosphate monobasic 4.0 grams
Sodium phosphate dibasic (anhydrous)
(pH should be 7.2 ± 0.5)
6.5 grams
  1. Place in fixative as soon as possible.
  2. Fix tissue slices (0.5 cm thick) 24 - 48 hours. Formalin volume must be 10 times or more than the volume of tissues.
  3. Use wide-mouthed, leak proof containers. DO NOT USE NARROW-NECKED CONTAINERS: A fresh and pliable tissue can easily be put into a narrow-mouthed container, but it becomes hard when fixed, and cannot be readily removed.
  4. Alternatively, tissues can be fixed in formalin solution for 24 - 48 hours, removed from solution, wrapped in formalin-soaked gauze sponge, placed in plastic bag, and sealed for transportation. This technique decreases the possibility of spillage and leakage of formalin during transportation.
  5. Improper handling or fixation of tissues can induce artifacts that may result in non-diagnostic or unsuitable specimens. A few examples that cannot be corrected by processing are:

a. Chemical dehydration - disinfectants and medications applied
    to the skin may damage (burn) tissues
b. Freezing - intracellular and extra cellular ice formation causes
    cell lysis and disrupts tissues
c. Pressure - excessive pressure from forceps or digits can
    rupture cells and compress tissues
d. Delayed fixation - un-refrigerated specimens are subjected to
    dehydration, autolysis and proliferation of saprophytic
    bacteria
e. Inadequate fixation - using inadequate volume of fixative or
    trying to fix large specimens (e.g. whole kidney or testis) will
    result in incomplete fixation and autolysis.

 

3. Writing a Post-mortem Report

The post-mortem report should record accurately and thoroughly all observations made during the examination of the carcass. A summary of the available history, clinical observations and results of field tests conducted must be included.

OBSERVE carefully and then DESCRIBE completely. Do not interpret! Theoretically, a person with a reasonably good command of a language will be able to describe well what that person has seen while performing the postmortem examination even though he/she does not know the significance of what he/she has seen and described! To help remember points that need to be described, remember this phrase: TeN DiSC SPaCeS

T - Tissue (eg. liver, colon)
N - Number (1, 2, too many to be counted)
D - Distribution (eg. diffuse, multifocal)
S - Shape (eg. round, oval)
C - Colour (eg. red, black)
S - Size (eg. 2 cm in diameter)
P - Pattern (eg. anteroventral)
C - Consistency (eg.soft, firm, hard)
S - Special features (eg. hemorrhagic exudates, swollen)
 


Use extra paper if the space in the standard form is insufficient and clip it together with the form.

Example: The liver is tan to brown and there are multiple 2 mm to 1 cm black foci on the surface and randomly distributed in the parenchyma.

Report negative findings only if relevant eg: absence of gross lesions in animal which had shown neurological signs

REMEMBER: For every diagnosis missed because you do not know about it, you will miss nine diagnoses just because you do not observe carefully!
 

  4. Hollow Organs

Organs such as intestine, urinary bladder, uterus should be cut open longitudinally (with care) to ensure fixation of mucosa. Sections of intestines should be fixed within a few minutes after removal.
   
  5. Solid Viscera

Organs such as liver, spleen and kidney should be cut into slices perpendicular to the surface to demonstrate their anatomic structure, whenever possible, one surface should consist of the natural boundary of the organ. Sliced tissue should be between 0.5 - 1.0cm thick to allow proper fixation. Please submit adequate samples and avoid small minute samples.

   
  6. Lesions Sampling

If lesion is small and localized, please include adjacent normal tissue as well as the lesion. This will help in identifying the tissue as well as defining the nature of spread of the lesions. In the case of large lesion, submit the entire mass with transverse cuts to allow formalin penetration, or if this is not feasible, submit appropriate sized sections from several different areas.

   
  7. Samples from a necropsy

Submit sections of abnormal tissue and organs. Clinical history and gross pathological findings may help in the selection of tissue samples for histopathological examination. One of the most common problem for the pathologist involves the receipt of a history and/or gross description that indicates the probability of lesions in specific organ or organ system, along with several tissues, none of which originate from the involved organ! This happens often with animals showing CNS signs, as there seem to be some reluctance to remove the brain and/or spinal cord. Another common example is a case of diarrhea with no submission of faecal or gastrointestinal tract samples. In cases where there are no clinical history, no gross lesions or sudden death, a complete set of organs should be collected. The recommended set of organs include:

Tissues with lesions
Brain
Heart
Lungs
Liver
Spleen
Kidney
Stomach
Small intestines
Large intestines
Lymph nodes
Urinary bladder
Thymus (young animals)
Bone marrow
Muscles
Adrenal glands
Thyroid glands
Other tissues in certain cases

REMEMBER: WE LOSE NOTHING IF WE TOOK MORE TISSUE SAMPLES
                    THAN WE ACTUALLY NEED, BUT WE MAY LOSE EVERY
                    THING IF THE TISSUES REQUIRED FOR FURTHER TESTS
                    HAVE BEEN THROWN AWAY!!!

   
  8. Brains

Brains submitted for pathological examination may be submitted intact and immersed in 10 times or more volume of 10% formalin. Better fixation and therefore examination can be obtained by prefixing the entire brain for two days in a large container in 10% formalin and then shipping the entire brain in a smaller container with a small volume of 10% formalin. This method is preferred.

   
  9. Skin biopsies

Skin biopsy samples require careful selection and handling. Do not shave biopsy sites as the hairs are useful guidelines for proper plane of section. Select recent active lesions and margins of lesions incorporating normal skin. If a bullous skin disease is suspected, the active lesion i.e. an actual blister is required for definitive histological diagnosis.

   
  10. Heart submission

The heart may be submitted in its entirety, particularly if a malformation is suspected. Rinse the chambers with water and fill with 10% formalin.

   
  11. Other helpful information

It is always wise for the pathologist to freeze and hold portions of organs, so that further tests such as virology, bacteriology, toxicology, etc. may be carried out later if necessary. So, please submit bigger portions of organs in ice to the laboratory.

   
  AVIAN SUBMISSIONS
 
 

In order to provide you with the most reliable test results, we have established criteria for specimens submitted to the Regional Veterinary Laboratories. We request your assistance in providing quality specimens.
 

  A. Serum samples - To be shipped

Submit at least 15 sera/group. A group = a house, flock. Do not pool samples or make up a group with sera from more than 1 house or source.

  1. Whole blood specimens should be allowed to clot, the serum micro centrifuge tubes.
  2. Each group of specimens should be put into a plastic bag and labeled appropriately.
  3. Pack the samples in an insulated container on dry ice or freezer packs and ship to the laboratory within one day. About 4.5 kg of dry ice or 6 - 10 freezer packs are required. These are minimum quantities to adequately chill the specimens. The specimens must arrive at the laboratory within 36 hours of collection.
  4. Do not ship whole blood specimens, please.
  5. Sera to be tested for Mycoplasma are not to be frozen. Put these specimens with freezer packs only in the Styrofoam container. Do not use dry ice.
  6. Serum to be tested for antibodies to agents other than Mycoplasma may be frozen.

B. Serum samples - To be brought in to the laboratory

  1. The specimens may be prepared as in para A1 and A2. These may be refrigerated up to 24 hours before transporting to the laboratory.
  2. Freshly drawn whole blood may be submitted if they are submitted on the same day as drawn. Older specimens tend to hemolyze or contaminate.

    a. These need to be collected in covered vials, snap-cap culture tubes,
        or vacutainer tubes.
    b. Please do not collect in open tubes.
    c. The blood should be allowed to clot on a 5 degrees slant. This
        allows maximum yield of serum. Do not allow blood to clot forming
        a 'plug' in the bottom of the tubes.
    d. If there is any delay in getting the samples to the lab, the clotted
        tubes may be refrigerated temporarily. Please do not put them in car
        trunks or anywhere that may overheat.

C. Tissue specimens for virus isolation - To be shipped

  1. Tissues submitted for virus isolation should be collected as aseptically as possible and put in plastic bags. All the air is expressed from the bag and the bag is closed securely.
  2. The plastic bags are placed into a small light-weight cardboard box which is in turn packed on dry ice in a Styrofoam shipping container. This prevents the tissues from being 'burned' direct contact with dry ice. This method requires 4.5 kg dry ice for transit times of 36 hours or less. Cool packs may be used in place of dry ice. Use at least 6 packs for overnight transit.
  3. We must insist on shipment by overnight express (e.g. Federal express, Nationwide or any other courier services).

D. Tissue specimens for virus isolation - To be brought in to the laboratory

  1. Tissue are collected and placed in plastic bags as in para C1 and labeled.
  2. Place the labeled plastic bags on ice and transport to the laboratory.

E. Bacteriological specimens - Shipped or brought in

  1. Obtain specimen using a 'culturette'. This is a pre-packed sterile swab which has a small ampoule of liquid media at the bottom. Be sure to contact only the material to be tested. Be sure to crush the ampoule immediately.
  2. Ship labeled culturettes at room temperature. Please do not refrigerate or freeze.
  3. Send culturettes by overnight express or any appropriate method so as to arrive at the laboratory in less than 24 hours from the time the specimen was obtained. Swabs in transit longer than 24 hours are almost always overgrown disproportionately or fail to yield the desired isolate.

F. Tissues for histopathological examination - To be shipped or brought in

  1. Tissues should be collected and put promptly into 10% buffered formalin. The tissues must not constitute more than 10% of the combined tissue/formalin volume. Pieces of tissue larger than 1 cubic centimeter should be partially cut to permit rapid penetration of formalin to the interior portions.
  2. Permit tissues to fix for 24 hours, drain excess formalin and reseal the container. This will save on the shipping charges; or
  3. The formalin need not be poured off and the tissues shipped as soon as they can be put into the formalin in suitable containers.
  4. Do not freeze any tissue intended for histopathological examination whether they are fresh or fixed.
  5. Pack the specimens in a leak-proof container with absorbent material and seal.
  6. Ship as expeditiously as possible.
  7. Be sure to label all specimens.

G. Necropsy

  1. Domestic poultry - Necropsy. When birds are submitted for necropsy, it is advantageous for you to:

a. Submit 4 - 5 freshly dead birds, that have died of the
    condition, preferably refrigerated and kept cool until they
    arrive to the laboratory. Time is important as small carcasses
    decompose quickly.

b. Submit 4 - 5 live, sick birds, that are clinically affected with
    the condition. Take care not to select cull birds from the flock
    which do not represent the disease condition. These
    specimens should be shipped by express in containers
    suitable for live birds.

c. If dead birds have to be submitted then try to bring the
    freshest specimens possible.

d. Please supply flock history, medication, vaccination, or any
    other information that may be helpful.

  1. Exotic or pet birds - necropsy. More often than not these birds have already died. Dead birds should be wetted and refrigerated immediately in a plastic bag. Package the wet bird in a plastic bag and ship to the laboratory on ice so as to arrive in less than 24 hours if possible. If this is impossible, freeze the bird and ship as suggested. However, we cannot perform histopathological examination on previously frozen tissue, only gross pathology, virus isolation, and bacteriology are possible.

 

  Guidelines for specimens for certain types of problems

Respiratory problems

Histopathology: Trachea, lung, air sac, nares, maxillary sinus, Harderian glands, bursa, thymus
Sera: 20 acute and 20 convalescent samples
Virus isolation: Trachea, respiratory tract, caecal tonsils
Bacteriology: Trachea, lung, air sac, liver, spleen
   
  Enteric diseases
 
Histopathology: 3 small intestine sections - duodenum (with pancreas), jejunum (near Meckel's diverticulum) and ileum (near ileo-caecal junction). Place each portion in a separate labeled container. Cecum, bursa, thymus, proventriculus and gizzard
Sera: 20 acute and 20 convalescent serum samples
Virus isolation: Caecal tonsils and any affected organs
Bacteriology: Caecal tonsils and any affected organs
   
  Leg problems
 
Histopathology: Hock, shank, tendon, synovial membrane, heart, foot pads (check - if not normal, submit), nerves - sciatic and celiac plexus (near top of kidney), muscle adjacent to sciatic nerve. Bone - must slit open before putting into formalin.
Sera: 20 acute and 20 convalescent samples
Virus isolation: Tendon, cloacal swabs, caecal tonsils
Bacteriology: Swabs of synovial fluids
   
  Central nervous system
 
Histopathology: Brain (cerebrum and cerebellum with the brain stem), proventriculus, gizzard, pancreas, neck, trachea, air sac and lungs
Sera: 20 acute and 20 convalescent samples
Virus isolation: Brain, trachea, air sac, lung, caecal tonsil, cloacal swabs
Bacteriology: Lung, air sac, liver, spleen and affected organs
   
  Production problems
 
Histopathology: Reproductive tract including ovaries, trachea, lung, air sacs, liver, kidney, spleen, bursa, thymus, caecal tonsils
Sera: 20 acute and 20 convalescent samples
Virus isolation: Reproductive tract, trachea, lung, air sac, caecal tonsils, cloacal swabs
Bacteriology: Liver, spleen, reproductive tract, caecal tonsil, air sac, bone marrow
   
  Suggested Standard Submissions from Aborted Fetuses
   
 
SPECIES HISTOPATH. BACTERIOLOGY SEROLOGY
Bovine Lung Lung Paired sera
  Heart Spleen  
  Liver Cotyledon  
  Kidney Abomasal contents  
  Spleen Pericardial fluid  
  Adrenal    
  Cotyledon    
       
Porcine Heart Lung Paired sera
  Lung Spleen  
  Liver Placenta  
  Kidney Stomach contents  
  Spleen Pericardial fluid  
  Placenta    
       
Ovine & Caprine Lung Spleen Paired sera
  Heart Lung  
  Kidney Placenta  
  Spleen Abomasal contents  
  Cotyledon    
       
Equine Thymus Spleen Paired sera
  Lymph nodes Lung  
  Spleen Placenta  
  Heart Stomach contents  
  Lung    
  Liver    
  Kidney    
  Placenta    
   
  COMMENTS
  1. Histopath: Fix 0-5 - 1.0 cm thick portions of tissue in 10 volumes 10% formalin per volume tissue. Do not freeze, if at all possible.

  2. Bacteriology: Submit tissues in plastic bags labeled "BACT", put placenta in separate bag: fluids should be submitted in labeled plain (red top) tubes; these specimens should be refrigerated but not frozen.

  3. Serology: Collect serum at the same time of abortion and 2-3 weeks later; remove serum from clot and store frozen at your office; paired sera can then be submitted as indicated. Never freeze clotted whole blood with the expectation of using it for serology later as this will interfere with serological tests.

Suggested Standard Submissions for Neonatal Scours

HISTOPATH BACTERIOLOGY VIROLOGY PARASITOLOGY
Duodenum Jejunum Jejunum Ileal mucosal smear
Mid-jejunum Ileum Ileum Feces
Ileum Ileal and jejunal Fecal smear
Colon smears    
Heart      
Lung      
Liver      
Kidney      
Spleen      
   
  COMMENTS:
  1. HISTOPATH: Fix 0.5 - 1 cm thick portions of tissues in 10 volumes of 10% formalin per volume of tissue; do not freeze; either open intestine longitudinally or fill lumen with formalin and ligate both ends.

  2. BACTERIOLOGY: Submit tissues in separate, labeled plastic bags. Smears for Gram stain may be made from a composite ileal/jejunal swab (not the sections for culture) and air-dried.

  3. VIROLOGY: Tissues may be submitted frozen, clearly labeled "VIROLOGY".

  4. PARASITOLOGY: To make ileal mucosal smears, transfer ileal mucosal scrapings to a glass slide and air dry. Fecal material should arrive at the laboratory in refrigerated condition (not frozen) as soon as possible after the sample is taken. Alternatively, feces may be preserved in 2.5% potassium dichromate solution (1 volume feces to 1 volume potassium dichromate solution).

  PARASITOLOGY & HEMATOLOGY
 
  1) Fecal samples
 
  Fecal samples should be fresh and preferably collected from the rectum (and not from the ground) so that the identity of the animal established. At least 10g of fecal material is required for each examination. The samples should be collected in a plastic bag or bottle, sealed, labeled and dispatched in ice to the laboratory. In cases where the fecal sample will take longer than 24 hours to reach the laboratory, submission in 10% formalin is required to prevent the parasite eggs from hatching. The volume of 10% formalin added should be approximately the same as the volume of feces submitted.
Tests: Fecal egg count, sedimentation for flukes, coccidiosis
   
 

2) Blood (Heparin/EDTA)

Hematology is the first step in the diagnosis of a disease or condition so blood samples should be labeled with the animal identification and sent to the laboratory as soon as possible (if travel time to the laboratory takes more than 6 hours it should be packed in ice) for hematology and blood protozoa examination. On collection, blood should be gently mixed to prevent clotting. For the preparation of thin/thick blood smear refer to page ___. Blood samples for determining trypanosomiasis (Surra) MUST be packed in ice and sent to laboratory within 24 hours of collection otherwise the organism will die and cannot be identified.
Tests: CBC, Blood protozoa e.g trypanosomes, Babesia.

 
3) Skin scraping
 
  Deep skin scrapings should be taken from the edge of the lesion. Scrape with a sharp blade sufficiently deep to cause slight bleeding over the area. A drop of glycerine or any oil may be smeared on the blade or skin area to make the skin scraping stick. Submit in a bottle or sealed plastic bag without additional preservative.
Tests: Identification of mites, etc.
   
  4) Parasitological specimens

a) Ticks, mites, lice, fleas, flies, snails and maggots should be collected and preserved in 70%  alcohol for identification.

b) Nematodes such as round/hook worms from the GIT, heart, should be washed in normal saline gently and dropped into hot 70% methylated spirit. The worms may be left in this solution and submitted in a leak proof container for identification.

c) Tapeworms for identification should be submitted with their heads and mature segments intact. To collect the head, leave the worms that are still attached to the intestine in normal saline and carefully tease out their heads, then place worms in water bath at about 40-50oC for 10-30 mins. to kill the worm in the extended state. Transfer to 70% ethylated spirit and pack in leak proof bottle to be sent to laboratory.

d) Flukes should be washed in normal saline and gently pressed between 2 glass slides bound together with a rubber band. Leave in 10% formal-saline for 24 hours, then remove the slides and return the flukes to the 1% saline, put in a leak proof bottle and send to laboratory for identification.
 

  5) Submission of organ samples

Intestines from poultry should include the caecum for examination of coccidia. GIT from ruminants, felines, canines should be tied at both ends (esophagus & rectum) before sending to the laboratory. Other organs such as liver, lungs, heart, muscle etc. for leucocytozoon examination should be fresh and transported in ice chest to obtain best results.
   
 

PREPARATION OF THIN AND THICK BLOOD SMEARS

Collection of blood for preparation of films

It may be necessary to first clip the hair. Puncture the vein with a needle or pricker. The first few drop of blood should not be used as they are always contaminated and rich with platelets. Only a small drop of blood is required as it is almost impossible to prepare a film that is too thin. A very common fault is to use too much blood. A drop the size of the head of a pin is sufficient. From carcasses, blood from the cut end of an ear may be used.

   
  Preparation of blood films

It is important that all glass slides used are perfectly clean, free from scratches, grease, oil, thumb prints, etc. Grease can be removed with a clean cloth (or thoroughly-washed, clean, dry handkerchief) dipped in methylated spirit. The surface of the slide should not be touched with fingers but handled by the edges. The slides may be kept dust free by wrapping them in sheets of clean paper.

Technique for preparation of thin blood film

a) A clean slide is placed on a level surface and a very small drop of blood is deposited near the end of the slide leaving sufficient space for labeling. (see Fig.1 below)

b) Spread the blood in an even film by means of another slide (spreader). The spreading edge of the spreader must be perfectly smooth (free of chips)

c) i) The spreader is held between the thumb & index finger and drawn
       backwards towards the drop of blood. (See fig.2 below)

   ii) As it makes contact, the blood will spread along the edge of the
      spreader, which is then moved forward in a smooth gliding manner. (See
      Fig.3 below). The blood is therefore pulled behind the spreader and
      never pushed in front of it.
The angle at which the spreader is held
      determines the thickness of the blood film. Generally a 30o - 40o angle is
      satisfactory.

d) Wave the slide in air to dry it.

e) The slide must then be identified by the name of the animal or its number on the same side as the smear.

A thin blood film should consist of a single layer of blood cells and its margins should not reach the edges of the slide. Thin blood films should be taken in the shade if possible or they may dry too quickly and the red blood cells may become cremated. For the same reason slides should not be left in the sun before use.

Some common faults in the preparation of blood smears and their causes are tabulated below:

Faults Causes
Smear has spots without blood Greasy slide
   
Smear too short Drop of blood taken in step (a) was too small. Angle of spreader with slide too wide
   
Smear too long or too thick Drop of blood taken in step (a) was too big. Angle of spreader with slide too acute
   
Smear streaky, not of uniform thickness Spreader has irregular edge, slide support and push through movement was not even and continuous
   
  BACTERIOLOGY
 
  SUBMISSION GUIDELINES
  Submission of samples for bacteriology
 
  • Tissues should be as fresh as possible
  • Take samples from edge of lesion, and try to include tissue
  • Be careful to collect in aseptic fashion
  • Collect samples prior to antibiotic treatment
  • Submit generous portions of tissue or several millimeters of pus, exudates or feces
  • Maintain most samples at refrigeration temperatures (40C) rather than frozen en route to laboratory.
  Anaerobic Cultures
 
  • Good collection method is essential; exposure to air for more than 20 minutes can be detrimental

  • Animals dead longer than 4 hours are usually unsuitable

  • Tissues and liquid exudates are recommended (anaerobic bag or tube would be helpful)

  • Swabs are not acceptable unless shipped in anaerobic containers (special collection devices with reduced oxygen environment)

  Milk
 
  • Collect milk in sterile snap cap or screw cap tubes
  • Cool samples before submitting to the laboratory, and mail with ice packs. Samples may also be frozen without altering recoverability of pathogens.
  Blood
 
  • Not normally used for recovery of animal pathogens because bacteremia is intermittent. Call the laboratory for recommendations.
  Urine
 
  • Collect by cystocentesis, catheter or mid-stream catch.
  Skin lesions
 
  • Pustules. Disinfect surface with alcohol, allow to dry and aspirate material with syringe and needle.
  • If ringworm is suspected, pluck hair from lesion and scrape edge of lesion. Submit hair, skin scrapings, and scrap material.
  Fecal
 
  • Clean anus/cloacae from feces with clean water and collect sample from the rectum or cloacae with sterile swabs or rectal pinch.

  SELECTION OF TISSUES FOR MICROBIOLOGY
 
 
Disease Suspected/ Condition Causative Agent Tissue to submit
Abortion, bacterial Brucella sp.
Campylobacter sp.
etc.
Fresh whole fetus
Fresh whole fetus
Placenta with cotyledons, lungs and liver
Uterine discharge
Fetal stomach contents
Abortion, Mycotic Aspergillus sp. etc. Placenta with cotyledons
Fetal stomach contents
Actinomyces infection Actinomyces pyogenes
(Corynebacterium pyogenes)
Fresh material from excised contents
Actinomycosis, bovine Actinomyces bovis Material from abscess or swab or in syringe
Anaerobic infections Bacteroides sp.
Fusobacterium sp.
Eubacterium sp.
Biopsies & exudates in sterile syringe with plugged needle. Anaerobic transport systems (swabs, bags)
Anthrax Bacillus antracis Blood sample taken from superficial ear vein.
Swine: swabs from exudates and cut hemorrhagic lymph nodes. State on submission from that case is anthrax suspect
Arthritis Streptococcus sp.
Actinomyces pyogenes
Haemophilus sp.
Erysipelothrix
Mycoplasma sp.
Chlamydia
Entire joint in smaller animals. Joint swabs in transport media or joint fluid in sterile syringe
Atrophic rhinitis of swine Bordetella bronchiseptica
Pasteurella multocida
Clean external nares with alcohol. Swab deep into nasal cavity. Entire noses if animal has died
Blackleg Clostridium chauvoe
Clostridum novy
Clostridium septicum
Fresh piece of muscle with lesion. Section of muscle with lesion. Impression smears
Botulism Clostridium botulinum Food suspected of containing toxin. Section of fresh intestine, tied off. Large section of liver. Serum
Brucellosis Brucells ovis, etc. Aborted fetus, stomach contents, placenta, mammary lymph nodes & milk sample
Chlamydial infections Chlamydia psittaci Abortions-Affected cotyledon, vaginal swabs, fetal lung and liver.
Arthritis: aspirated synovial fluid.
Conjunctivitis: conjunctival swab. Samples should be submitted in transport media
Campylobacteriosis Bovine & Ovine Campylobacter fetus Fetus, cervical mucus, preputial washing semen. Deliver within 5-6 hours of collection. Freeze specimens and transport on dry ice. Transport medium can be obtained from laboratory. Fresh rectal/fecal swabs. Feces
Corynebacterial Pneumonia of foals Rhodococcus equi (Corynebacterium equi) Tran tracheal aspirate, nasal discharge swab, lung and lymph nodes with lesions, fresh
Cystitis Escherichia coli
Proteus sp.
Streptoccus sp.
(group D, fecal)
Coli forms
Fresh urine in sterile syringe or swab in transport media
Dermatitis Staphylococcus hyicius, aureus intermedius, Pseudomonas sp.
Streptococcus sp.
Fungi (Dermatophytes)
Clean surface to remove dirt. Swab of lesions in transport media. Fresh sections of skin
Dermatomycosis Microsporum sp.
Trichophyton sp.
Plucked hair and skin scrapings sent dry in envelope. Fresh & formalized sections of skin
Dermatophilosis Dermatophilus congolensis Scabs, crust & plucked hair. Skin biopsy after scab removal, fresh and formalized
Enteric disease Escherichia coli
Other coli forms
Salmonella sp.
Tied off affected sections of intestine. Swabs from intestinal sections
Enterotoxaemia Clostridium perfringens Several ounces of fresh intestinal contents in tube or bag. Cool & transport rapidly to laboratory. If interested in preserving toxin, freezing of ileal contents is preferred
Erysipelas Erysipelothrix rhusiopathiae Acute form: Heart blood, portion of kidney, spleen & liver
Arthritic & cardiac form: Swabs from affected tissues, heart valves
Exudative Epidermitis Greasy Pig Disease Staphylococcus hyicus Clean skin with soap, water and alcohol. Scrape lesions and send to lab on swab in transport media or in sterile syringe
Glasser's Disease Haemophillus parasuis Preferable a live, sick pig. Swabs for culture from various serous membranes & meninges
Johne's Disease Mycobacterium Para tuberculosis Ileocecal valve & mesenteric lymph nodes or scrapings from mucosa. Rectally collected fecal sample
Leptospirosis Leptospira sp. Darkfield exam on fluids & urine. Kidney tissue for FA
Listeriosis Listeria monocytogenes Neural form: Brain stems, fresh & formalized.
Visceral form: Liver, fresh & formalized.
Abortion form: Fetus & placenta
Lymphadenitis Corynebacterium pseudo tuberculosis
Streptococcus sp.
Fresh pus on swab from several lesions
Abscess tissue & lymph
Malignant edema Clostridium septicum Dead animal: Muscle and two smears from lesion.
Live animal: Fluid or material from lesion in a syringe
Mastitis Staphylococcus sp.
Streptococcus sp.
Mycoplasma sp.
Coli forms
Many other bacteria
Clean & dry udders. Strip several streams of milk before starting collection. Collect 1-2 streams from each quarter into sterile snap cap or screw cap tubes. Refrigerate immediately & maintain at 40C until cultured. If culture work is not performed within 24 hours, samples may be frozen up to 2 weeks
Meningitis Streptococcus sp.
Streptococcus suis
Haemophilus somnus
Cryptococcus neoformans
Cerebral spinal fluid
Brain
Mycoplasma infections Mycoplasma sp. Refrigerate and deliver hours of collection within 24-48 hours. Samples can be frozen & shipped frozen. May include mucosal scrapings, tracheal exudates, and aspirates. Lung tissue with bronchi, joint fluids and mastitis milk swabs should be submitted in transport medium
Otitis Externa Proteus sp.
Pseudomonas sp.
Staphylococcus sp.
Streptococcus sp.
Yeast (Pityrosporum sp.)
Ear swabs for bacterial & fungal culture
Pasteurellosis Pasteurella sp.
Mycoplasma sp.
Other bacteria
 
Nasal swab in transport medium/saline. Transtracheal washing. Portion of affected lung
Pleuro pneumonia of swine Actinobacillus pleuropneumoniae Portion of lung with lesion
Proliferative ileitis   Fresh portions of affected intestine including spiral colon & ileum
Pyelonephritis, Bovine Corynebacterium renale Midstream sample of urine. Portion of affected kidney, ureter, bladder & urethra
Salmonellosis Salmonella sp. Rectally collected feces, fecal swab in animal showing signs.
Dead animal: Intestine, liver, spleen, lung & lymph nodes
Strangles Streptococcus equi Fresh pus taken on a swab from an excised abscess
Swine dysentery Serpulina hyodysenteriae Live, sick pig for necropsy. Fresh material from colonic mucosa. Tied off section of spiral colon
Trichomoniasis Trichomonas sp. Preputial wahsing. Vaginal mucous
Tuberculosis, Bovine Mycobacterium bovis Smears for acid fast stain
   
  MYCOLOGY
 
  For the isolation of dermatophytes, infected and apparently healthy hairs at the periphery (edge) of the lesion should be submitted. The hairs may be plucked with forceps or a pair of tweezers. Deep scrapings of skin from the edge of the lesion may also be submitted. The specimens are placed in clean screw-capped bottles or envelopes, packed and submitted as such without preservation. Scabs from suspected cases of streptothricosis should be collected early in the disease.
   
  For systemic mycosis, send specimens from affected organs in ice. For culture of moldy feed sample, at least 50 gms. should be submitted.
   
  GUIDELINES FOR THE SUBMISSION OF SAMPLES FOR SEROLOGICAL TESTS AND THE INTERPRETATION OF TEST RESULT
 
 
TEST INDICATION FOR TEST FREQUENCY AND NO. OF SAMPLES TO BE SUBMITTED INTERPRETATION OF TEST RESULT
A) Bacterial Disease
1) Brucella abortus
   
Brucella abortus CFT (Cattle, Buffalo, Pig, Goat, Horse) 1) Herd test to monitor incidence of disease in farms; to evaluate control and eradication program; Herd Health program; to screen replacement or newly imported animals All eligible animals in hers once every 4 months unless otherwise indicated. Cattle and buffalo - Females 12 months and above (If unvaccinated) or 24 months and above (if vaccinated). Males for breeding - 6 months and above. Other animals - sexually matured
Negative Doubtful Positive
Vaccinated cattle/buffalo
<1/4 2/4-3/8 >4/8
Unvaccinated cattle or animals of unknown vaccination status
<1/2 2/2 >2/4
Bulls and bull calves
Other animals

 

  2) Clinical Cases - abortion, stillbirth, premature birth, retained placenta, and infertility. In horses: fistulas withers, poll evil Paired serum from affected and immediate in contact animals 1. Animals doubtful to the test should be re-test 2 months later. If doubtful on 3 consecutive occasions, then animals should be considered positive.
2. Sera of animals that are anti-complementary (A/C) should be resubmitted at once. If result found to be A/C for 3 consecutive tests, animal should be considered as positive
Brucella melitensis CFT (Sheep and goats only) Herd Health program; newly imported animals and for transfer out of farm and before distribution All eligible animals in farm once every 4 months or otherwise indicated
Negative Doubtful Positive
<1/2 2/2-1/4 >2/4
Animals that are positive should be culled immediately
Brucella ovis CFT Herd test as in Br.melitensis esp. male animal. All imported animals, animals for transfer out of farm and herd health program and small holder's animals. Once a year or otherwise indicated. All breeding males and females
Negative Doubtful Positive
<1/10 2/10-3/10 >4/10
Clinical cases: Epididymitis, infertility and abortion Affected animals
Brucellosis Rose Bengal Plate Test (RBPT) for cattle, buffalo, goats and pig Interstate movement and animals in quarantine station that have to go out early before CFT result can be obtained All eligible animals. Test can be carried out at point of entry by an authorized personnel or send to the nearest laboratory Positive: Isolate animals and branded with B+.
Negative: Submit sera for Brucellosis CFT confirmation test.
Note: Until otherwise informed, all sera whether positive or negative should be submitted for CFT
Brucellosis Milk Ring Test (BMRT) for cattle, buffalo and also goats milk Dairy Herd Test used as a screening test and as a monitoring test in eradication programme. Done by Milk Collecting Center (MCC) All MCC should submit bulk milk or churn or pail samples every month Positive: Submit all animals in the group to the standard blood test
Brucellosis CFT: Isolate animals until CFT result is available.
Negative: Group of animals tested can be considered free of disease and regular testing with BMRT must be continued from time to time to ensure clean dairy herds
 
Brucella canis 2 Mercaptoethanol tube agglutination test (2ME - TAT) Dogs for export. Dogs for breeding All breeding age dogs. Breeding animals should be tested twice a year Interpretation of titers will depend on type of antigen used. The testing laboratory will report as positive, doubtful or negative
Clinical cases: abortion, infertility Affected animal and sire or bitch
Johne's disease CFT for cattle, buffalo, goats and sheep Herd test as in Brucellosis CFT. Newly imported animals, animals newly introduced into a farm and animals belong to small holder farmers. NOT DONE on all animals already in farms if already clean of disease. All eligible animals above 12 months (cattle and buffalo) or 6 months (goats) of age once every year Titers 3/10 and above is considered significant. Send fecal samples from such animals for microscopic and culture examination. Animals should be considered positive if they fulfill one of the following criteria:

1. Culture positive with CFT significant or Not significant.
2. CFT =/>3/10 and AFB positive
3. CFT =/>3/10 and good clinical evidence of Johne's disease
4. CFT =/>3/10 on repeated tests.

Clinical cases - wasting, emaciation, intermittent diarrhea Affected animals - repeat testing every month if necessary
Leptospirosis Microscopic Test (LMAT) Test is only done on clinical cases whereby paired serum are required and not as a screening test.
Clinical cases - Haemoglobinuria, jaundice, intermittent pyrexia, wasting, abortion, infertility and sudden drop in milk production
Paired sera from affected animals 1. Titers of 1/100 and above are considered significant.
2. When a sample has significant titers to more than one sero group, the sero group with the highest titer is considered more important.
3. When there is a 4-fold or more increase in titer in a convalescent serum, it is indicative of recent or current infection
Melioidosis CFT (All species) Not done as a routine screening test.
Clinical cases: orchitis, epididyrmitis, pyrexia and abscessation
Affected animals - repeat testing may be necessary
Negative Doubtful Positive
<1/4 1/4-4/4 =/>1/8
All doubtful animals should be re-test within 2-3 weeks later
Caseous lymphadenitis Gel Diffusion Test (GDT) (Sheep and goats) Not as a screening test for animals in farm. Herd health program.
Clinical cases:
Enlarged lymph nodes (parotid, sub mandibular, retropharyngeal, pre scapular, pre femoral, supra mammary, popliteal), fever if systemic, respiratory problem, anorexia, wasting, drop in milk production
Affected animals and immediate in contact animals. Repeat testing may be necessary
  GDT Clinical exam. Action
Goats +
+
+
-
Cull
1. Isolate & re-test if good body condition
2. Cull if poor body condition
- + Cull
Sheep +
-
-
+
Cull
Cull
Note: Positive - Isolate animals & re-test after 1 month
GDT: Cull if positive on 2 or more testing
Salmonella dublin SAT 'O' antigen (for cattle sera) Herd test: monitor extent of infection in an affected herd, monitor imported or newly introduced animals 20% of herd
Negative Doubtful Positive
<1/20 1/40 >1/80
Note:
1. Doubtful samples should be repeated 2-3 weeks later
2. From positive animals, send feces, milk, vaginal discharge, joint fluid (aspirated aseptically) for bacterial culture
Clinical cases - abortion, metritis, respiratory, enteric or joint disease in calves. Paired serum from affected animals
Check for possible "carrier" animals Repeated testing of recovered animals
Salmonella dublin 'H' antigen SAT (for cattle sera) Same as for 'O' antigen SAT Same as for 'O' antigen SAT
Negative Doubtful Positive
=<1/40 1/80 =>1/160
   
  VIROLOGY
 
  SUBMISSION GUIDELINES
 
  Submission of samples for virology
 
  • Submit specimens collected early in the course of the disease as this enhances of isolating the virus
  • Aseptic technique should be used in collecting materials
  • Swabs should be placed in test tubes containing 50% glycerol saline
  • All materials should be refrigerated (below 40C) immediately
  • Deep freezing is recommended for all materials without 50% glycerol saline
  • Where vesicular disease (foot and mouth disease) is suspected, it is important to notify the laboratory nearest to you. Do not proceed to collect specimens. Proper authority will do specimen collection and submission.
  SUBMISSION OF SPECIMENS ACCORDING TO SPECIFIC MAMMALIAN DISEASES
 
 

Disease

Source of specimens

Type of specimens to be submitted and preservative

In ice flask. Specimens should be in plastic bags preferred packed separately In 10% formalin (unless otherwise stated) to be submitted at room temperature Others at room temperature
Aujeszky's Live animal Paired sera As in ice Affected live piglets
Dead animal f1 brain, spinal cord, visceral organs, lymph node
Canine distemper Dead animal   Brain, stomach, kidney, lung  
Malignant catarrhal fever Live animal Un-clotted blood Nil
 
Fixed thin blood smear
Dead animal f1 brain, lung, heart, liver, kidney, mandibular and bronchial lymph nodes as well as rib f1 brain, lung, liver, spleen, kidney, eye, intestine and lymph nodes Fixed thin blood smear if carcass is fresh
Rabies Dead animal f1 brain in glycerol-saline f1 brain in Zenker's acetate solution Nil
Swine fever Dead animal
(If a dead animal is not available, sacrifice one sick animal for examination and in addition, submit samples from live animal as indicated
f1 brain, loop of intestines, lung, liver, spleen, heart, kidney, mesenteric, tonsil, pancreas and portal lymph nodes as well as rib if carcass is autolysed or contaminated during post-mortem examination f1 brain, lumbar, spinal cord and other organs as stated for submission in ice except for rib Nil
Live animal
(If possible, collect specimens from untreated animal. Otherwise, proceed with collection of specimens and of treatment in your submission form)
Paired sera   Affected live piglets, indicate type fixed thin blood smear and un-clotted blood
Transmissible gastroenteritis Live animal Paired sera Small intestines from 3 levels, namely duodenum, jejunum and ileum, separately and labeled Affected live piglets
Dead animal Entire gastrointestinal tract
   
  SEROLOGY/VIROLOGY
 
 

With the large volume of serology cases, it is very important for samples to be submitted in such a way that they can efficiently be logged into the laboratory.

 
SUBMISSION GUIDELINE
 
1. Send serum only, not whole blood or clots and allow 0.5ml serum per test
    requested (1 ml minimum). Procedure for obtaining good quality serum:

    a) Always use dry needles, containers, and tubes
    b) Do not freeze blood before collecting serum
    c) Do not force blood through needles
    d) Do not use outdated corvac tubes

2. Place serum in plastic snap cap tubes. This will aid efficient setup and
    storage, saving time in getting the results back to you. If you use blood
    tubes with silicone plugs, also put the serum in plastic snap tubes. This
    allows us to store the sera for up to 30 days following testing.

3. Number the tubes consecutively, 1 through x, matching the actual tube
    number with the tube number written on the serology submission form.
    Use permanent marker to identify tubes. Grease pencil marks are easily
    rubbed off, and labels fall off during the heating process necessary for
    some tests.

4. Keep cases from different owners separate.

5. Make a copy of the paperwork submitted, and retain a portion of each
    serum sample in your freezer. This guards against un-forseen mishaps
    (lost or broken tubes) and may save you from having to re-bleed. Acute
    serum samples may also be retrieved from the freezer for pairing with
    convalescent samples. Paired sera should be tested together to
    maximize the diagnostic value of test results.
 

  GUIDELINES FOR THE SUBMISSION OF SAMPLES FOR SEROLOGICAL TESTS AND THE INTERPRETATION OF TEST RESULTS
 
 
Test Indication for test Frequency & no. of samples to be submitted Interpretation of test results
1. Avian encephalomyelitis (AE) - embryo susceptibility test
   
  40 fertile eggs of age less than 3 days old are submitted per flock This test is carried out to determine the immune status of a parent flock and its progeny against AE
Avian encephalomyelitis (AE) - AGP test 30 sera or more per flock should be submitted for the test A positive test indicates exposure to the AE virus
2. Aujeszky's disease - SN test Clinical cases:
Older animals -  nasal discharge, dyspnea, ataxia, pyrexia, dullness, anorexia
Sows, gilts - reproductive failure
Young piglets - nervous disorders with high morality
Herd test - indicates prevalence only
 
Uncontaminated paired sera for diagnosis SNT titer of 8 or above considered significant
4-fold rise in titer in convalescent serum indicates recent or current infection
3. Bovine viral diarrhea (BVD) - SN test (cattle, buffalo) Clincal cases - inflammation of mucosa, abortion, malformed fetuses, chronic debilitation, fever, rapid respiratory rate, fulminate diarrhea, mummification of fetus Uncontaminated paired sera for diagnosis SNT titer of 10 or above considered significant, but negative titer could indicate "carrier" status
 
4. Duck virus hepatitis (DVH - SN test (duck)   Pooled serum samples from 30 or more affected ducks between the ages of 1 day to 6 weeks should be submitted This test is strain specific. A serum neutralization index (SNI) of >2 is considered positive for DVH antibodies
5. Egg drop syndrome (EDS) - HI test (poultry)   30 sera or more per flock should be submitted for the test. Paired (acute & convalescent) sera are preferred. This test is carried out only for disease investigation HI titer of >1/2 is considered positive. This can be interpreted as an exposure to EDS virus
6. Equine herpes I-SN test (horse)   Individual serum samples from horses are to be submitted. Uncontaminated serum samples from all ages HI titer of >2 is considered positice. This can be interpreted as an exposure to EHV type I
7. Equine infectious anemia - Coggin's  test (horse)   Individual serum samples from horses are to be submitted. A minimum of 8 sera should be submitted A positive test indicates exposure to the EIA virus
8. Infectious bronchitis (poultry) IB-AGP test   30 sera or more per flock should be submitted for the test A positive reaction indicates an exposure to the virus due to vaccination or infection. This test is group specific
Infectious bronchitis IB-SN test 30 sera or more per flock should be submitted for the test A serum neutralization index (SNI) of >2 is considered positive for antibodies due to natural exposure or vaccination. This test is strain specific but not group specific
9. Infectious bursal disease (IBD) - AGP test (poultry)   30 sera or more should be submitted per flock for the test. This test is carried out only for disease investigation A positive reaction indicates an exposure to the IBD virus
10. Infectious bovine rhinotracheitis (IBR) - SN test (cattle, buffalo) Clinical cases - conjunctivitis, pyrexia, cough, dyspnea, increased & open-mouth labored respiration, anorexia, increased salivation, abortion, vulvo-vaginitis, weight loss, drop in milk production Uncontaminated serum samples from all ages. Uncontaminated paired sera for diagnosis SNT titer of 8 or above considered significant. 4-fold rise in titer convalescent serum indicates recent or current infection
11. Japanese encephalitis (JE) - HI test (horse, swine) Clinical cases - fever, jaundice, petechial hemorrhages, nervous disorder, difficulty in swallowing, stillbirth. Herd test - indicates prevalence only Uncontaminated serum samples from adults 4-fold rise in titer in convalescent serum indicates infection
12. Newcastle disease (ND) - HI test (poultry)   30 sera or more should be submitted per flock for the test HI titer of f1 is considered positive for antibodies due to natural exposure or vaccination
13. REO virus - AGP test (poultry)   30 sera or more should be submitted per flock for the test. This test is carried out only for disease investigation  
14. Swine fever - SN test (swine) Clinical cases - loss of appetite, sluggishness, dyspnea, diarrhea with tenesmus, high fever, conjunctivitis, irregular blotching of ears, legs & belly. Herd test - indicates vaccination or prevalence only Uncontaminated serum samples from all ages. Uncontaminated paired sera for diagnosis SNT titer of 8 or above considered significant. 4-fold rise in titer in convalescent serum indicates recent or current infection.
Note: It is useless to perform SNT for vaccinated animals

 

15. Transmissible gastroenteritis (TGE) - SN test (swine) Clincal cases - high mortality & morbidity of piglets with diarrhea. Herd test - indicates prevalence only Uncontaminated serum samples from adults or recovered piglets 4-fold rise in titer in convalescent serum indicates infection. SNT titer of 8 or above indicates recent or past infection
16. Western equine encephalomyelitis (WEE) - SN test (horse) Clinical cases - paralysis of lips, legs, depression, fever Uncontaminated serum samples from adults 4-fold rise in titer in convalescent serum indicates infection. SNT titer of 8 or above indicates recent or past infection
17. PRRS Clinical cases - still birth, respiratory problem Uncontaminated serum samples from adults ELISA (+VE/-VE)
   
  NOTE:
 
 

* Paired sera means acute & convalescent sera. The acute phase serum is collected at height of infection or at the time of clinical signs & the convalescent serum is collected 2-3 weeks later. Identify samples as A or 1 (for acute) & C or 2 (for convalescent). Preferably send both samples TOGETHER to the lab as paired sera. They may be sent separately but the reference no. of the acute or first serum must be stated when the convalescent or second serum is sent later.

   
  TOXICOLOGY
 
  SUBMISSION GUIDELINES
   
 

A thorough interchange of information through a written or telephone history between clinician and diagnostician is important for confirming diagnoses suspected by the attending clinician. A complete account of history (including management and feed type), symptoms, and lesions submitted with specimens for laboratory evaluation is very important.

All specimens and exhibits for toxicological examination are to be sent to the Chemistry Department directly or via the regional laboratory. MAKVET 06 form should be used for this purpose.

There is no practical or affordable way to check for all possible poisons. The more information provided to the chemist, the more efficient and useful would be the selection process.

Selecting specimens for toxicology and chemical evaluation require three main criteria.

1. The correct specimens must be provided based on the type
    of poison involved
2. Adequate amount of specimen must be available
3. Preservatives should not be used unless absolutely
    necessary.

  GUIDELINES FOR CHOICE AND CONDITION OF SPECIMENS
   
 
  1. Specimens should be free of chemical contamination and debris. Contamination of samples with hair, vomitus, dust, dirt, etc. may produce erroneous results.
  2. Freeze animal and tissue specimens for chemical analysis. Do not freeze whole blood samples, but keep them refrigerated.
  3. Serum, if separated from the clot and not hemolysed, may be frozen.
  4. Always package specimens from various organs and fluids separately.
  5. Use clean glass or plastic containers that can be tightly sealed.
  6. Label each specimen (container) for clear identification.
  7. Never add preservative such as formalin unless there is a specific reason. If preservatives are added, include a sample of the preservative along with the specimen.
  8. Submit samples for chemical analysis of low level organic contaminants, such as pesticide residues or PCB's, in glass containers, not plastic. Solid samples for this purpose may be wrapped in aluminium foil.
  9. If ammonia, urea, or cyanide toxicosis is suspected, freeze rumen contents and blood or serum immediately after collection. Keep them frozen.
  10. On all toxicology cases involving a dead animal, submit both fresh and formalin-fixed tissues.
  MAINTAINING PACKAGE INTEGRITY
   
  Integrity of a package must be maintained for legal or insurance purposes and proper sealing procedures followed.
   
  TISSUE SPECIMENS
   
  Analyses are directed toward exposure sites (skin and digestive tract), areas of metabolism and excretion (liver, kidney, urine), and accumulation in specific affected organs or storage sites (e.g. brain, fat, bone).

Specimens that should be submitted from a live animal include:

Serum (clot removed) 5 ml
Whole blood (in anticoagulant) 10 ml
Urine 50 ml
Vomitus or feces 200 g
Milk (if appropriate) 100 ml

Specimens that should be submitted from a dead animal include:
 

Serum or whole blood (if available) 10 ml
Urine 50 ml
Liver 100 g + formalin fixed
Kidney 100 g + formalin fixed
Body fat 100 g
Brain (f1 frozen, f1 formalin) divide by midline sagittal section
Rumen or stomach contents 200 g
Eyeball Whole eye
Bone 100 g
   
  FORAGE
   
 
  1. Sample size for hay and silage should be at least 1 quart. Cut all forages to a length of 3" or less.

  2. Pack samples tightly to exclude air, and seal airtight in plastic bag. Very dry sample may be submitted in paper bags.

  3. Take standing pasture and row crop samples for a minimum of 8-10 locations within a field. Remove forage from a four-foot area at grazing height. Mix all collected forage and take a representative sample (500 grams) for analysis.

  WATER
   
 
  1. Water samples should represent the water to be tested.

  2. Submit water samples for inorganic analysis in clean containers of either glass or plastic. Container should be sterile if microbiologic testing is requested.

  3. Submit all water samples for organic analysis in clean glass jars with clean aluminium foil placed over the mouth of the jar before attaching the lid.

 

QUICK GUIDE FOR TOXICOLOGY SPECIMENS

Specimens required for diagnosis of common toxicants are listed in Table 1.

Table 1: SPECIMENS REQUIRED FOR TOXICOLOGY TESTS

 
Poison or Analysis Specimen Required
Alkaloids stomach content, liver, urine
Ammonia whole blood, serum, rumen content
Anticoagulant rodenticides whole blood, liver, bait
Arsenic liver, kidney, urine
   
Carbon monoxide whole blood, fetal thoracic fluid
Carbonates whole blood, brain, rumen/stomach contents
Cholecalciferol feed, serum, kidney
   
Chlorinated hydrocarbon insecticides brain, liver
Copper liver, kidney, serum, feed
Cyanide stomach/rumen content, liver, whole blood, plant
   
Ethylene glycol fixed kidney, stomach content, bait
   
Gossypol feed, fixed heart
Herbicides stomach/rumen content, liver, feed, water
Monensin, Lasalocid feed, heart, skeletal muscle
   
Lead whole blood**, liver, kidney
Magnesium serum, eyeball
Metaldehyde stomach content, bait
   
Nitrate, Nitrite eyeball, rumen content, forage, water
Organophosphate insecticides whole blood, brain, rumen/stomach content
Oxalates
Petroleum/fuel  products fixed kidney, forag,e rumen/stomach content, lung
   
Poisonous plants rumen/stomach content, plant
Selenium liver, serum, feed
   
Strychnine stomach content, liver
Sulfonamides urine, kidney, feed (freeze)
Urea  
  ** EDTA (purple top) tubes are fine
   
  SPECIMENS FOR DIAGNOSIS OF SPECIFIC TOXICANTS

Information is presented in Table 2 for special considerations for individual chemistry analysis are required.

TABLE 2: SPECIMENS REQUIRED FOR SPECIFIC TOXICOLOGY TESTS

 
Poison or Analysis Specimen Required Amount of Specimen Comments
Alkaloids
(Caffeine, Nicotine strychnine theobromine)
Liver
Kidney
Stomach content
Urine
Feed/bait
100 g
50 g
100 g
10 ml
100 g
 
Ammonia

 

Whole blood serum
Rumen contents
Urine
5 ml
100 g
5 ml
 
Frozen (1-2 drops of saturated HgCl2 may be used instead of freezing rumen contents). 
Antibiotics Liver
Kidney
Stomach content
Feed
Urine
Milk
100 g
Whole
100 g
100 g
50 ml
200 ml
 
Contact the lab for details. Individual tests for penicillin, erythromycin, lincomycin, chloramphenicol, tetracycline, neomycin and tylosin.
Anticoagulant rodenticides Whole blood
Feed
Liver
Bait/other materials
5 ml
100 g
100 g
100 g
Includes brodifacoum, diphacinone, chlorphacinone, warfarin
Arsenical feed additives Liver
Kidney
Feed
50 g
50 g
250 g
Submit sciatic nerve and/or brachial plexus in formalin
Bromethalin Bait
Stomach contents
  Assault, Vengeance
Contact Lab
Carbon monoxide Whole blood, fetal thoracic fluid 10 ml  
Chlorinated hydrocarbon insecticides





Cholecalciferol
Brain (cerebrum)
Stomach contents
Rumen contents
Liver
Body fat
Kidney
Blood
Feed
Bait
Kidney (fixed)
Serum
Half of brain
100 g
100 g
50 g
10 g
50 g
10 ml
100 g
100 g
50 g
10 ml
Must not be contaminated with hair or stomach contents; preferable to place in chemically clean glass jars; avoid plastic containers, wrap specimen in clean aluminium foil
Cholinesterase Whole blood
Brain (refrigerated or frozen)
10 ml
Half of cerebrum
 
Cyanide Whole blood
Liver
Forage, silage
Skeletal muscle
Other materials
10 ml
50 g
250 g
100 g
100 g
Freeze specimens promptly in airtight container
Dicoumarol Liver
Rumen contents
Forages/feed
Blood
100 g
100 g
100 g
10 ml
 
Estrogenic agents Liver
Feed
Forage
100 g
100 g
100 g
Zearalenone, zearalanol, coumestrol, and diethylstibestrol
Ethylene glycol Serum
Stomach content
Bait
Kidney (in formalin)
Urine
10 ml
100 g
100 g

10 ml
 
Fluoroacetate (1080) Stomach contents
Liver
Kidney
Urine
Bait
All available Freeze biological specimens
Gossypol Feed 100 g Referred to outside laboratory
Herbicides (atrazine, paraquat, 2,4-D) Plants
Urine
Rumen contents
Liver
Kidney
Water
Soil
100 g
50 ml
200 g
50 g
50 g
100 ml
100 g
Contact lab
See also chlorinated hydro-carbons insecticides and organophosphorus or carbonate insecticides
Metals
Antimony
Arsenic
Urine
Kidney
liver
10 ml
50 g
50 g
 
Chromium
Cobalt
Copper
Iron
Lead
Mercury
Molybdenum
Nickel
Thallium
Zinc
Whole blood
(EDTA or heparin)
Feed
Serum (nor for lead)
10 ml
100 g
5 ml
 
Monensin, lasalocid Feed
Rumen contents
Heart (in formalin)
Skeletal muscle (in formalin)
200 g
200 g
Whole
Naracin, salimomycin
Mycotoxins (aflatoxins, zearalenone, vomitoxin, T-2 toxin, ochratoxins, citrinin, slaframine, ergot, fumonisins) Liver
Kidney
Stomach or rumen contents
Feed
Forage
100 g

100 g
200 g
200 g
200 g
200 g
 
Available individually or in a panel (see Table 3). Not all mycotoxins can be routinely analyzed in tissues
Nitrated or nitrites Water
Forage, silage
Eyeball
Rumen content
Other materials
50 ml
100 g
Whole
250 g
100 g
Keep refrigerated
Oxalates Fresh forage
Kidneys (fresh and fixed)
6-8 plants
Whole
One kidney in large animals, both in small animal
Phenols Stomach contents
Rumen contents
Other materials
550 g
500 g
500 g
Pack in airtight container
Submit in glass, not plastic
Pyrethroid insecticides Brain
Insecticide material
100 g
100 g
 
Selenium Serum
Whole blood
Feed
liver
Hair/hoof (chronic)
2 ml
10 ml
50 g
50 g
50 g
 
Sulfonamides Feed
Urine
250 ml
10 ml
 
Urea Feed
Other materials
100 g
500 g
All specimens should be frozen. See Ammonia
   
  SCREENING PANELS

Several analyses grouped by clinical or chemical family are available as screening panels. See Table 3.

TABLE 3: CHEMISTRY SCREENING PANELS

 
1. Canine CNS
    Nicotine
    4-aminopyridine
    Caffeine
    Strychnine

3. Organophosphorus Insecticides

5. Carbamate Insecticides

7. Fungicides
    Captan
    Carboxyl
    Thiram
    PCP
    PCNB
    Thiocarbamates

9. Mycotoxin Tremorgens
    Penitrems
    Aflatrems
    Fumitremorgens
    DAS

11. Herbicides
     Atrazine
     Simazine
     Bladex
     Sencor
     2,4 D

13. Sulfas
     Sulfamerazine
     Sulfa pyridine
     Sulfaquinoxaline
     Sulfathiazole
     Sulfadimethoxine

2. Plant Alkaloids
    Atropine & hyoscyamine
    Solanidine
    Coniine
    Scopolamine

4. Chlorinated Hydrocarbon
    Insecticides

6. Pyrethroid Insecticides

8. Hormonally Active/Feed
    Compounds
    Zearalenone
    M.G.A. (Melengesterol acetate)
    D.E.S. (Diethylstibesterol)

10. Mycotoxins
     Aflatoxins
     Zearalenone/Zearalenol
     Vomitoxin
     Ochratoxin
     T-2

12. Anticoagulant Rodenticides
     Warfarin
     Dicoumarol
     Diphacinone/Chloropacinone
     Brodifacoum

14. Water Quality
     Nitrate/Nitrite
     Total Dissolve Solids
     Sulfates
     Coli form bacteria


Each panel can be done as a group when history or clinical signs are not specific enough to suggest individual toxicant analysis.

   
  CLINICAL CHEMISTRY
   
 

Most clinical chemistry analyses require whole blood, plasma or serum. Always check the test protocol for the type of sample required.

For blood chemistry, enzymes, substrates and minerals are determined frequently.

Plasma samples can be refrigerated or frozen.

To avoid hemolysis, do not centrifuge clotted blood at speeds higher than 3000 rpm (700 x g) or for a prolonged time. Serum should be chilled immediately and transported in ice. Exposure to sunlight should be avoided.

Biochemical test combination kits e.g. wet chemistry kits from Boehringer Mannheim, Roche etc or dry chemistry slides from Idexx or Kodak are available commercially. Automated or semi-automated chemistry analyzers may be employed if the sample number is large.

Glucose concentration in blood can drop 10% an hour if kept at room temperature due to glycolysis. Use tubes with sodium fluoride (10mg/ml) or EDTA (2.5mg/ml) if plasma cannot be removed immediately by centrifugation.

Urine samples collected either by cystocentesis, catheterization or natural voiding should be chilled immediately after collection and kept chilled until arrival at the laboratory. Urine dipsticks which allow qualititative and semi-quantitative measurements may be purchased commercially.

Suggested profiles for tissue abnormalities and metabolic disorders is found in Table 1.

Sample handling before centrifugation and storage recommendations for serum or plasma after sampling is explained in Tables 2 and 3.

   
 

Table 1: Suggested profiles for tissue abnormalities and metabolic disorders

   
General profile Cardic profile
Albumin Albumin
Alkaline Phosphatase (AP) AP
Alanine aminotransferase (ALT) (GPT) ALT
Amylase Aspartate amonotransferase (AST) (GOT)
Calcium Creatinine
Cholesterol Creatinine kinase
Creatinine Lactate dehydrogenase (LDH)
Glucose Total protein
Phosphate Urea
Total bilirubin  
Total protein  
Urea  
Endocrine profile Hepatic profile
AP Albumin
ALT APALT
AST ALTAST
Amylase AST, Cholesterol
Calcium Cholesterol
Creatinine Gamma glutamyl trasferase (GGT)
Glucose LDH
Lipase Ammonia
Phosphate Total bilirubin
Triglyceride Total protein
Urea Triglyceride
Lipid profile Renal profile
Albumin Albumin
Cholesterol Amylase
Glucose Calcium
Total protein Glucose
Triglyceride Lipase
Phosphate
Total protein
Pancreatic profile Gastro intestinal abnormalities
AP Albumin
ALT Amylase
Amylase Calcium
Calcium Glucose
Cholesterol Lipase
GGT Phosphate
Glucose Triglyceride
Lipase Total protein
Phosphate
Triglyceride
Urea
 
Dietary effects Drug-induced alterations
Albumin AP
AP ALT
AST AST
Calcium Cholesterol
Cholesterol Glucose
Creatinine GGT
Glucose LDH
Phosphate Magnesium
Total bilirubin Total bilirubin
Total protein
Triglyceride
Urea
 
   
 

Table 2: SAMPLE HANDLING BEFORE CENTRIFUGATION FOR USE WITH VETTEST 8008

 
TEST Delay in centrifugation Blood sample must be centrifuged immediately Comments
ALB
Albumin
OK    
AP,
Alkaline phosphatase
OK    
ALT,
Alanine aminotransferase (GPT)
OK    
AMYL,
Amylase
OK    
AST,
Aspartate aminotransferase (GOT)
  YES Slight hemolysis can cause marked increases in plasma AST activity
Ca2+
Calcium
OK   Avoid exposure of sample to the air
CHOL,
Cholesterol
OK    
CK,
Creatinine Kinase
  YES Slight hemolysis can cause marked increases in plasma CK activity
CREA,
Creatinine
OK    
GGT,
Gamma-glutamyltransferase
OK    
GLU,
Glucose
  YES In lithium heparin glycolysis occurs in the presence of red cells; the glucose concentration can diminish at the rate of up to 10% in an hour at 200C
LDH,
Lactate dehydrogenase
  YES Slight hemolysis can cause marked increases in plasma LDH activity
LIPA,
Lipase
OK    
MG2+
Magnesium
  YES Hemolysed samples can give erroneous high magnesium concentrations
NH3,
Ammonia
  YES Avoid exposure of the sample to the air. All sample containers should be capped unless sample is being withdrawn, to ensure that loss of ammonia or contamination does not occur
PHOS,
Inorganic phosphate
  YES Phosphates are released quickly from the red cells. Hemolysed samples can give erroneous high phosphate concentrations
TBIL,
Total bilirubin
  YES If immediate analysis is impossible, the plasma must be removed and stored in the dark between 4 and 80C as bilirubin degrades rapidly in light
TP,
Total protein
OK   Hemolysed samples can result in raised plasma protein concentrations
TRIG,
Triglycerides
OK    
UREA/BUN,
Urea
OK    
   
 

Table 3: STORAGE RECOMMENDATIONS FOR SERUM OR PLASMA AFTER SAMPLING

 
Constituents +200C to +250C (Room temp) 0 to + 40C (Refrigerator) -200C (Freezer) Comments
Alkaline Phosphatase (ALKP) Loss of activity 10% after 7 days 7 days 7 days Levels may increase; if frozen, thaw & left at 40C
ALT (GPT) 2 days 7 days Unstable Is not stable during thawing
AST (GOT) Loss of activity 10% after 3 days Loss of activity 8% after 3 days 7 days  
Bilirubin, total Use only fresh serum     Do not expose to sunlight
Calcium 10 days 10 days 6 months Cork stopper increases Ca level
Cholesterol, total 6 days 6 days 6 months  
Cholinesterase 7 days 7 days 3 months  
Copper 14 days 14 days    
Creatine Kinase Loss of activity 2% after 7 days Loss of activity 2% after 3 days Unstable Analyze ASAP
Gamma-GT 2 days 7 days 1 month  
GLDH Loss of activity 5% after 3 days Loss of activity 2% after 3 days 7 days  
Glucose 1 hour 4 hours 3 days Deproteinise immediately
Iron 4 days 7 days    
Magnesium 1 week      
Phosphorus, inorganic 2 days 7 days 10 days  
Protein, total 2 days 7 days 10 days  
SDH     2 days Assay immediately
Urea 1 day 3 days 6 months  
Uric acid 8 hours 7 days 2 months  
   
  RABIES SUBMISSION
   
  A. Pre-handling procedures

All carcasses of rabid animals must be handled with care so as to avoid exposure. Individuals involved with handling of rabid specimens should be adequately vaccinated against rabies and properly attired (lab coat or coveralls, plastic apron, thick rubber gloves, a mask, cap, rubber boots and goggles are a must). Ensure that any unauthorized personnel are not in the vicinity.

B. Submission Form

All details about the rabid animal must be entered into the submission form (MAKVET 5A). Ensure that the form is completely filled. All dog-bite cases must be clearly stated in the column provided to differentiate routine submissions. Each sample (head) should be properly labeled and accompanied by a form (one head one form).

C. Specimen Preparation

If the clinical history suggestive of rabies, extra precaution should be taken. The suspected rabid animal should be euthanised before decapitation. Avoid damaging the head if it has to be shot so that there is minimal brain damage. Extra precaution should be taken to prevent contact with saliva and other body fluids. The following procedures should then be followed:

(i)  The head should be decapitated at the atlanto-axial joint. Decapitation should be carried out on a solid surface or table so that it is easy for disinfection.

(ii)   The head should be cooled down promptly and kept cold (40C). Freezing should be avoided. [Note: Although freezing the specimen in dry ice during transit or in liquid nitrogen flasks will preserve the virus, a prompt microscopic examination may be delayed because of the time taken to thaw the specimen].

(iii)  All openings that include the mouth, nostrils and ear orifices should be stuffed with formalinised cotton or cloth to prevent spillage of fluids.

(iv)  Each head should be put into a suitable watertight container and closed tightly. If plastic bag is used, choose the thicker one and make sure that it is sealed or tied properly with a rubber band. This in turn should be put into a larger watertight container, where cracked ice is packed between the inner and outer container. The package should be clearly labeled (given Bag No.) and accompanied by the completely filled MAKVET 5A form. The package is then put into a specially designed metal chest and shipped to the Veterinary Research Institute, Ipoh, Perak with the fastest possible courier. The metal chest should be labeled as below:

AWAS! Bahan Biologik
MERBAHAYA
Jika bekas Rosak
Sila Telefon Segera 05-5457166/87
Institut Penyelidikan Haiwan, Ipoh

[Bahan perlu disimpan pada 40C]

   
  GUIDE TO SUBMISSION OF SPECIMEN ACCORDING TO MAIN CLINICAL SIGNS OBSERVED (MAMMALIAN CASES ONLY)
   
 
Main Clinical Problem Source of Specimens

Type of specimens to be submitted and preservative

In ice flask In 10% formalin (unless otherwise stated) to be submitted at room temperature Others at room temperature
Abortion (Note: Samples from dam must always be included) Fetus (Important: Please see remarks in 1st column before proceeding) Abomasal/stomach contents (or preferably intact abomasum/stomach with both ends tied), 1/2 brain, liver, lung, intact kidney, spleen, lymph nodes, also skin if there are any lesions - for instance in the case of mycotic abortion Pieces of the same organs as stated for specimens to be submitted in ice (including the other half of the brain) NIL
In the case of fetuses, you may either submit the specimens as indicated in column 3 & 4 Placenta (fetal membranes) Piece of placenta including cotyledons depending on species of animals NIL Fixed thin blood smear
Dam-alive Serum samples (Collect two samples; at time of abortion and resubmit 2-3 weeks later) and vaginal, cervical or uterine swabs/washing
OR
you may send the entire fetus in ice as an alternative
Dam-dead Portion of uterus and oviduct tied off. Visceral organs including iliac and other regional lymph nodes if systemic disease suspected Pieces of ovary, oviduct uterine wall, cervix and vagina, as well as visceral organs if systemic disease suspected NIL
Abscesses Affected organ or tissue Swab of pus or intact abscess collected in a sterile manner Wall of abscess and adjoining unaffected tissues NIL
Anemia Live animal Serum sample, un-clotted blood Fecal sample in 10% formalin* Fixed thin blood smear, fecal sample** and urine
  Dead animal Spleen, lung, kidney, liver, brain Spleen, lung, lymph node, liver, bone marrow (in separate bottle) or a rib, kidney and brain. Also fecal sample in 10% formalin* Air-dried impression smears of spleen, lung, lymph node, liver, bone marrow and brain. Fecal sample** and fixed blood smear
Dermatitis Live or dead animal Scabs if present Scabs if present and skin biopsy if possible Deep skin scraping in sealed bottle without any preservative
Diarrhea and Dysentery Live animal Rectal scraping or rectal swab. Un-clotted blood in fluoride vials and urine from calves, sheep and goats suspected of enterotoxemia. (Note: The blood and urine are required for sugar tests. If dip reagent "stix" are available, results from these dip tests would be sufficient), Fecal sample in 10% formalin for long distance transport, i.e. if specimens will take more than 24 hours to reach the lab. Fecal sample (from areas close to the lab.)  i.e. if specimens will reach the lab. within 24 hours
  Dead animal Loop of affected portion of intestine including mesentery with lymph nodes attached (tie off both ends of intestine), lung, liver, spleen and kidney. In the case of calves, goats and sheep suspected of enterotoxemia, send intestinal contents with some chloroform added to make up 1%, and sample of urine as stated for live animal Piece of rumen, abomasum/stomach, intestine, lymph nodes, liver, spleen, kidney and brain from calves, sheep or goats suspected of enterotoxemia. Note: In young pigs - if there is concurrent vomition with diarrhea, include sections of duodenum, jejunum, ileum (each in separate bottles labeled accordingly) and lumbar spinal cord in addition Fecal sample. If it will take more than 24 hours for the fecal sample to reach the lab. submit in 10% formalin. Air-dried smear of contents of colon from cases suspected of swine dysentery
Emaciation Live animal Serum sample, un-clotted *Feces in 10% formalin Fixed blood smear, smear of rectal scraping gently fixed over flame (ruminants only), feces**
  Dead animal As many organs as possible including brain, (and portion of ileum, ileocecal valve and part of cecum from cattle) Organs as listed for submission in ice and *feces in 10% formalin  
Hematuria and Hemoglobinuria Live animal Un-clotted sera and paired NIL Urine and fixed blood smear
Dead animal Brain, liver, lung, heart, spleen, kidney, lymph nodes and urinary bladder As listed for submission in ice Urine and fixed blood smear. Fixed impression smears of brain, lung, liver, spleen and kidney
Infertility (This laboratory can only handle infectious causes of infertility. Since this requires special investigation, you are advised to contact the A.I. service and thelab.) Female animal - live Serum samples, cervical or vaginal mucus NIL Contents of uterus in sterile bottle
Female animal - dead or killed Serum samples (if possible), portion of ovary, oviduct, uterus, cervix and vagina Portions of reproductive tract as stated for specimens to be submitted in ice
Male animal -dead or killed Pieces of testicle, epididymis, accessory sex glands, (prostrate, seminal vesicle and bulbo-urethral gland depending on species of animal) and portion of penis if abnormal As stated for specimens to be submitted in ice NIL
Jaundice Live animal Serum sample, urine, fecal sample Fecal sample in 10% formalin (for long distance transport, i.e. if specimens will take more than 24 hours to reach the lab.) Un-clotted blood, fixed blood smear, *fecal sample, serum and urine
  Dead animal Liver, kidney, feces and urine Liver, including cause of obstruction of bile duct if any, kidney, spleen, lymph nodes, bone marrow in separate bottle or long bone. Fecal sample in 10% formalin Fecal sample**, urine, serum if possible and fixed blood smear
Joint  swelling (Arthritis) Live animal Joint fluid provided strict aseptic technique can be used NIL NIL
Dead animal Intact joint and if there is any systemic signs of disease, include brain, lung, spleen, kidney and lymph nodes Joint capsule (including synovial membrane) and other organs as listed for submission in ice
Mastitis Live animals Milk sample from all quarters - label samples according to quarters NIL NIL
Dead animal Portion of affected udder and supra mammary lymph node As for in ice
Metritis Live animal Vaginal discharge in sterile bottle. If there is no discharge, submit uterine, cervical or vaginal swab/washing in that order of preference. Serum sample - repeat 3 weeks later NIL Fixed thin blood smear. Un-clotted blood
Dead animal Portions of uterus and fallopian tubes tied off. If there is extension of inflammation unto the urinary system, include a swab of the contents of the urinary bladder and one intact kidney especially in cases of chronic metritis. In the case of sows, include a piece of mammary gland as well if any particular quarter shows evidence of concurrent mastitis Ovaries, oviducts and pieces of uterus (include shreds of fetal placenta if present). If urinary system is secondarily involved, submit portions of urethra, urinary bladder and kidney as well. Also in the case of sows, include a piece of mammary gland from affected quarter if there is concurrent mastitis NIL
Nervous disorders (Specimens for SWINE FEVER are indicated previously) Dead animal Portions of brain, spinal cord, liver, lung, tonsils, and mandibular lymph nodes Brain, spinal cord, liver, spleen, heart, urinary bladder, kidney, lung and stomach. (The brain and spinal cord should preferably be fixed in  Bouin's in suspected cases of Aujeszky's disease. If Bouin's solution is not available, use 10% formalin NIL
Orchitis (See also infertility) Live animal Aspirated exudates, if any, from scrotal sac or testis using sterile technique, serum sample NIL  
Dead animal Affected testis, accessory sex glands and any other affected organs as well as regional lymph nodes As for specimens to be submitted in ice NIL
Peritonitis (Other than traumatic reticulo- peritonitis) Live animal Aspirated abdominal exudates provided that asepsis can be ensured NIL  
Dead animal Abdominal exudates, liver, spleen, kidney, lungs, heart and 1/2 brain As for specimens to be submitted in ice except for abdominal exudates NIL
Poisoning Dead and/or live animal Liver, kidney, spleen and stomach contents and wall of stomach or rumen and abomasum Brain, spinal cord, lung, heart, liver, spleen, kidney, stomach (or rumen or abomasal) wall, small and large intestines, lymph nodes and possibly bone marrow in a separate bottle 1. Thin blood smear air dried or fixed in methyl alcohol
2. Un-clotted blood in McCartney bottle.
3. Suspected source of poisoning e.g. feed, bait, dead rodents etc.
4. Intact plants including roots or representative portions of plants
Respiratory problems Live animals Un-clotted blood from buffaloes, nasal discharge or nasal swab NIL Send specimens as for hemorrhagic septicemia (thin blood smear, air dried)
Dead animal Turbinate, larynx, trachea and lung. If no lesions in these organs, send brain and piece of diaphragm (if affected) and as many of the other organs possible, including normal looking lung, mandibular, bronchial and mediastinal lymph nodes Lung, lymph nodes, brain, rumen, liver, heart, spleen, kidney and piece of diaphragm (if affected) NIL
Septicemia (Whenever possible, specimens should be collected from an untreated animal. If treatment has already been instituted, proceed with collection of specimens and indicate type of treatment given in the submission form) Live animal Un-clotted blood sample NIL NIL
Dead animal 1/2 brain, lung, liver, heart, spleen, kidney and loop of small intestines. If carcass is too decomposed or contaminated during post-mortem examination, submit rib (select one that would fit the flask) in addition As for in ice, except for rib If carcass is fresh, submit fixed blood smear
Tumor Live or dead animal Biopsy, post-mortem material Pieces of the tumor from every location including transitional zone with surrounding normal tissue NIL
Water belly (ascitis) and dependent edema Live animal If asepsis can be ensured, submit a few ml of aspirated abdominal fluid in sterile bottle *Fecal sample in 10% formalin Fecal sample**
Dead animal Abdominal fluid or exudates. Heart, including valves if lesions are present As above and pieces of abomasum, intestines, heart, including valves, liver, spleen, kidney and lung. In the case of small animals, especially cats, check ovaries etc. for tumors, and if present, submit several pieces As above. Abomasal and intestinal parasites in 70% ethyl alcohol
UNCERTAIN CASES AND SUDDEN DEATH WITHOUT ANY APPARENT GROSS LESIONS Dead animal As many organs as possible including brain. Un-clotted blood and serum if possible As many organs as possible including brain. *Fecal sample in 10% formalin Fecal** sample and fixed blood smear
  *  If specimens will take more than 24 hours to reach the lab.
** If specimens can reach the lab. within 24 hours
   
 

Poultry Cases

 
Disease Specimens required for diagnosis Remarks
  1. 3-5 birds showing early signs of
   disease
Send by fastest possible transport. Pack birds in boxes with water-proof bottom and label accordingly
  2. 3-5 birds in advanced stage of
   illness, or dead birds
 
  3. 3-5 birds which are apparently
    healthy
Boxes should have holes to allow ventilation
  4. Affected organs from dead birds
    examined in the field. These
    specimens must be submitted in
    10% FORMALIN
Dead birds in plastic bags in ice flasks. Organs collected in the field should be in 10% temperature (not in ice)

 

BAGI SEBARANG PERTANYAAN LANJUTAN

Sila hubungai : -
BAHAGIAN KESIHATAN VETERINAR
Jabatan Perkhidmatan Veterinar Negeri Selangor
Lot 2, Jalan Utas 15/77,
40630 Shah Alam,
Selangor.


Tell : 03-5510 3900
Fax : 03-5510 3903

 Kawalan Penyakit    |    Drug & Vaksin


 [ Laman Utama ]