Methods of proper collection and dispatch of the microbiological materials for disease diagnosis
MANDEEP SHARMA
HOD
INTRODUCTION
Samples may be taken from animals, or their environment, for the purpose of establishing a disease diagnosis, for health surveillance, or for the monitoring of response to vaccines. In order to get timely and correct diagnosis of a suspected infectious disease, it is imperative on the part of a clinician to collect the most suitable material from live or dead animals. A great variety of different combinations of samples and species of animal may occur. The knowledge of pathogenesis of infectious disease is single most important factor in order to collect the most suitable specimen. In the face of an outbreak where animals in various stages of the clinical disease may be seen, it is better to collect specimen from fresh cases of the disease. In all cases, the samples need to be appropriate for the purpose required, and adequate in number and amount to provide a statistically valid result. Samples must be taken with care, to avoid undue stress or damage to the animal or danger to the operator. For example, a carcass suspected of being infected with Bacillus anthracis should not be opened, but a drop of blood should be obtained from a superficial vein. It is usually important to adopt aseptic techniques, and care must be taken to avoid cross-contamination between samples. Just prior to death and shortly thereafter, a number of intestinal bacteria may invade the host tissues. The significance of these organisms, some of which are potential pathogens is difficult to assess when tissues have been invaded. For best results, fresh tissues must be collected as soon as it is feasible. Live sick animals presented for necropsy, invariably provide the best source of samples/specimens. For microbiological investigations, strict sterile precautions must be observed meticulously while collecting and handling materials for isolation studies. It requires at least as much effort, and often more, to process a negative specimen as it does one from which microorganism is isolated. The chance of isolating a microbe depends critically on the knowledge, care, and attention of the veterinarian who collects the specimen. Specimens taken as a last resort when days or weeks or empirically chosen antibiotic therapy have failed are almost invariably a waste of effort.
Having obtained suitable material, it must be carefully packaged, labeled, and transmitted to the laboratory by the fastest practicable method. Relevant shipping regulations must be obeyed. If material is sent to a laboratory in another country, this laboratory must be consulted in advance to ensure that it is willing to receive the material. An import license may be required. All samples must be accompanied by a written note indicating the origin of the material, the relevant history, and the tests required.
A. Equipment required for collection of samples
(1). Sterile forceps, scissors, and scalpels. (2). Sterile swabs (3). Vials for containing transport medium for collection of samples for isolation or identification (4). Bottles for collection of faeces, blood, and other samples that do not require transport medium (5). Bottles containing formalin saline for tissues to be examined histologically. (6). Blood collection equipment- without additive for serum, and with anticoagulant for isolation (7). Notebook and equipment for labeling specimens (8). Swabs and transport medium for bacteriological investigation (9). Cool box (Thermos flask) (10). Heavy duty plastic bags for postmortem material.
B. COLLECTION OF SAMPLES
1. Tissues (in general):
Animal health personnel should be trained in the correct procedures for post-mortem examination of the species of animals with which they work. The equipment required will depend on the size and species of animal, but a knife, saw and cleaver will be required, and also scalpel, forceps and scissors, including scissors with a rounded tip on one blade, for opening intestines. A plentiful supply of containers appropriate to the nature of the sample required must be available, and also labels, and report forms. Special media may be required for transport of samples from the field. The operator should wear protective clothing: overalls, rubber gloves and rubber boots. If rabies is suspected, it is usual to detach the animal's head, and the operator should wear a face mask and goggles, gloves and a plastic apron.
Tissues may be collected for culture or for histopathology and occasionally for use as antigen in serological tests. The person removing the tissues should be experienced in post-mortem technique and have knowledge of pathology sufficient to select the right organs and the most promising lesions for sampling. The skin of the dead animal may be removed with ordinary instruments, but the body cavities should be opened with sterile instruments, and a fresh set of sterile instruments should be used to collect the pieces of the various organs required. Each piece of tissue should be placed in a separate sterile screw-capped jar or plastic bag, fully labeled with the date, tissue and animal identification. Care must be taken not to contaminate one tissue with another. Instruments can be heated on a burner with portable packs of liquid gas or by using local fuel to light a fire. Disinfectants must not be used on or near tissues to be sampled for bacterial culture or virus isolation.
The fresh samples should be forwarded to the laboratory by the fastest direct route. If they can reach the laboratory within 24 hours they should be forwarded in a wide-mouthed vacuum flask with wet ice. An alternative is to use polystyrene containers and chemical refrigeration bricks. Only if the samples are likely to take more than 24 hours to reach the laboratory, it is necessary to freeze the samples and send them in this state. The tissues may be sent to the laboratory dry or in bacterial or virus transport medium depending on the examinations required. For histopathology, blocks of tissues not more than 0.5 cm thick and 1-2 cm2 are cut and placed in neutral buffered 10% formalin, which should be at least 4 times the volume of the tissue sample. Samples for histology should not be frozen. For some procedures, e.g. rabies, larger portions of brain are required, some fresh and some in fixative, and for Scrapie and BSE whole brains may be required.
2. Blood:
Blood samples may be taken for hematology or for culture and/or direct examination for bacteria, viruses, or protozoa, in which case the blood is added to anti-coagulants such as heparin. They may also be taken for serology, in which case a clotted sample is required. A blood sample is taken, as cleanly as possible, by venepuncture. In most large mammals, the jugular vein or a caudal vein is selected, but brachial veins and mammary veins are also used. In birds, a wing vein (brachial vein) is usually selected. Blood may be taken by syringe and needle or by needle and vacuum tube (not easy in delicate veins but convenient in strong veins). Ideally the skin at the site of venepuncture should first be shaved (plucked) and swabbed with 70% ethyl alcohol and allowed to dry.
Whole blood samples can have antibiotics added to reduce bacterial growth, taking care that the antibiotics are chosen so as to avoid interference with the growth of the pathogens concerned. For samples with anti-coagulant and/or antibiotics, thorough mixing is necessary as soon as the sample has been taken. It may be also necessary to make a smear of fresh blood on a microscope slide. For serum samples, the blood should be left to stand at ambient temperature (but protected from excessive heat) until the clot begins to contract. The clot can then be ringed round with a rod and the bottles then placed in a refrigerator at 4º C. Later, the serum can be decanted or removed after centrifugation. Chemical preservatives, such as boric acid or merthiolate, should be avoided in sera to be used in virus neutralization tests. An alternative method is to transport a drop of dried blood on a filter paper disk that contains enough material for sensitive antibody assay systems.
Whole blood samples are very good virological specimens for following diseases:
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Disease Preservative for blood
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Blue tongue OCG, Sod. Citrate, heparin
African horse sickness OCG
Rinderpest Heparin
Equine rhinopneumonitis Heparin
Equine infectious anaemia EDTA
Swine, fever EDTA
Marek's disease Heparin
Malignant catarrhal fever EDTA (1 mg/ml of blood)
3. Faeces:
Freshly voided faeces should be selected, and sent with or without a transport medium. An alternative and sometimes preferable method is to take swabs from the rectum (or cloaca), taking care to swab the mucosal surface. Swabs may also be transported either dry or in transport medium. Faeces for Parasitology should fill the container to reduce air and prevent hatching of parasite eggs.
4. Skin:
In diseases producing vesicular rashes or where lesions are exclusively in the skin, samples are taken from the lesions themselves. Scrapings of the lesion may be taken, and additionally the vesicular fluid should be sampled where unruptured vesicles are present.
5. Genital tract:
Samples may be taken by vaginal or prepucial washing, or by the use of suitable swabs. Sometimes the cervix or urethra is also sampled by swabbing.
6. Eye:
A gentle swab of the surface of the conjunctiva is taken and is broken off into transport medium. Scrapings may also be taken onto a microscope slide. Metal-handled swabs are useful to ensure sufficient cells are removed for microscopic examination.
7. Nasal discharge (saliva, tears):
Samples may be taken by soaking cotton swabs that are wetted with transport medium and sent to the laboratory at 4º C.
8. Milk:
Samples of milk should be taken after cleansing the tip of the teat. The initial stream of milk is discarded and a tube filled with the next stream(s). In severe mastitis, there may be little fluid present.
9. Environment:
Samples may be taken to monitor hygiene or as part of a disease inquiry, for example, from litter, ventilation ducts, feed troughs, drains, soil, hatcheries and slaughter houses.
10. Serum:
Serum samples are the most commonly collected specimens from live animals for conducting various serological tests. Generally serum samples early in the course of disease (acute, within 1-4 days) and during convalescence (convalescent, around 21 days) are collected. Such samples are called paired serum samples and are used to demonstrate the rising antibody titre. In comparing the antibody titres of acute and convalescent phase sera, a minimum four fold rise is considered significant.
C. SELECTION OF SAMPLES
Considerable skill and care are required to decide on the correct samples to be sent to the laboratory. Frequently a combination of blood samples for serology and tissues from dead or culled animals for microbiological culture will be required. Also, it is usually important to collect tissues for fixation for histology.
D. SAMPLE SIZE
There are some general statistical rules which should be borne in mind, particularly when sampling herds or flocks for a health surveillance scheme. It is possible to calculate how many animals must be sampled from a herd/flock of a certain size, to achieve a 95% probability infection assumed to be present in a certain percentage of the animals.
E. INFORMATION TO BE SENT WITH SAMPLES
Information and case history should always accompany the samples to the laboratory, and ideally should be placed in a plastic envelope. The information should include the following points:
1. Name and address of owner/occupier where disease occurred, with telephone and fax numbers,
2. Disease suspected,
3. Samples submitted and tests required (transport medium used),
4. Different species on the farm and number, age and sex of each affected animal,
5. Length of time on the farm; if recent arrival, where from,
6. Date of first cases and of subsequent cases or losses,
7. Description of the spread of infection in the herd or flock,
8. Number of animals dead, the number showing clinical signs, and their age, sex and breed,
9. The clinical signs and their duration including the condition of mouth, eyes and feet, and milk or egg production data,
10. Type and standard of husbandry, including the type of feed available, possible contact with poison or poisonous plants,
11. A list of description of the samples submitted for examination, and post-mortem findings,
12. Any medication already applied to the animals, and when given,
13. Any vaccination already given, and when given,
14. Name and address of sender, with telephone and fax number, and date of submission.
F. TRANSPORT OF SAMPLES
Samples must be carefully packed, to avoid any possibility of leakage or cross-contamination. They should be delivered within 48 hours and must be kept cool during transit. Some samples should not be frozen. Screw-capped bottles should be used and should be additionally sealed with adhesive tape or paraffin wax. Samples in individually identified containers should be placed in larger strong, outer containers and packed with enough absorbent material to protect from damage. Official shipping regulations must be consulted. It is advisable to contact the laboratory in advance in the case of unusual requests. It is essential to do so, where material is sent to a laboratory in another country. Many countries require a special import license to be obtained in advance for any biological material, especially for tissues which could contain animal pathogens. This should accompany the package and be attached in an envelope to the outside of the parcel.
G. Preservation of specimens
Various preservatives are used for different specimens, e.g. phosphate buffered glycerin for tissues; EDTA, sodium citrate, heparin or OCG mixture for whole blood and transport media (TPB) for swabs. The preserved specimens are most frequently transported on ice in a thermos flask or other suitable containers.
A. Phosphate buffered glycerin (PBG):
It is prepared by mixing equal parts of phosphate buffered saline and neutral glycerin (pH 7.0).
Phosphate buffered saline (PBS)
Sodium chloride (Nacl) 8.00 g
Pot. chloride(Kcl) 0.20 g
Di-Sod. hydrogen phosphate 1.15 g
(Na2 HPO4)
Pot. dihydrogen orthophosphate 0.20 g
(KH2 PO4)
Glass Dist. water 1000 ml.
The final pH of PBG sol. is adjusted between 7.2 to 7.4, before autoclaving.
B. Oxalate-Carbolic acid-Glycerin (OCG) Mixture:
Potassium oxalate 5.0 g
Phenol (Carbolic acid) 5.0 g
Glycerin 500 ml
Dist. water 500 ml
The pH is adjusted to 7.2 before autoclaving.
C. Tryptose phosphate broth (TPB):
It is used as a transport medium for nasal, eye, rectal swabs etc.
Tryptose (Difco) 20.0 g
Dextrose 2.0 g
Sod. chloride 5.0 g
Di-sod. hydrogen phosphate 2.5 g
Dist. water 1000 ml.
The pH is adjusted to 7.2-7.4 before autoclaving. Antibiotics (Penicillin, Streptomycin, Mycostatin) are added before collection of swabs to check bacterial contamination.