Introduction

Specimen collection is part of a disease investigation especially when you for various reasons want to know the cause, rather than treat based on a clinical examination

Disease investigation in animals usually follows a similar pathway:

1. Sick animal/animals
2. Collection of background data (so-called “history”) on the animal/s
3. Owner/carer observations (part of history taking)
4. Distance observation of animal/animals
5. Clinical examination of the animal/s
6. In-practice tests as indicated i.e. haematology; urine examination; blood biochemistry (often indicates the organ system involved); ultrasound; radiographs
7. Samples for in-house and, if necessary,  for laboratory testing
8. Diagnosis
9. Control the disease

At each step, you will be defining problems and listing the most likely causes in order of likelihood known as a differential diagnosis. Some of the differential diagnoses that you have listed will be easily excluded in the early stages of your disease investigation, others may require additional testing. For this, you will have to collect specimens either for in-practice tests or to be sent away to an appropriate laboratory for an aetiological (cause) diagnosis.

When do you want an aetiological diagnosis? (That is, know the cause)

1. When a large number of animals are affected i.e. communicable disease, poison
2. When the treatment is dependent on knowing the cause
3. To determine a prognosis
4. Diseases that are unresponsive to empirical treatment i.e. antibiotic resistance
5. Unusual disease events i.e. increased abortion of cows on a farm that has not had this problem before
6. Diseases having a marked effect on animal production and welfare
7. Serious Zoonosis
8. State notifiable disease
9. Animal welfare/Legal/Insurance cases – chain of evidence
10. Academic – When you wish to publish case studies.

Relevant laboratory results can only be provided if specimen collection has been done well.

• A good knowledge of the disease pathogenesis including the possible aetiology agents will assist you in the selection of the correct animal, specimen/s and sampling site.
• Know the more common diseases in your work environment.
• However, still be on the lookout for unusual disease events. These are the ones that can have serious consequences and you would not be in the habit of recognising them.

When presented with individual animals, there is only one, so it would be the one to sample.

Tip: If you think there is a possibility that more testing will be required for a sick animal, but due to costs and other practical reasons, you decide to initially carry out empirical therapy, it is always worthwhile collecting an appropriate sample and storing it. It can always be discarded if not needed.

Ideally, one would want to sample clinically affected animals, that are untreated and in the early stages of the disease.

Key Takeaways

The best animals to sample in a disease outbreak are untreated animals in the early stage of disease.

 

Animals in the early stages of disease often show malaise (not feeling well/ are “off” and if inflammation is present they are feverish). In the case of highly transmissible diseases, it is valuable to sample at least 10% of the affected animal population. Include, in-contact animals as they may be either incubating the disease or in the early stages of the disease. and thus may have the aetiological agent present, but not complicating opportunistic/secondary pathogens.

Animals that have died provide you with the easiest means of sampling a wide range of tissues. However, they may not always be the best to sample as they may represent treatment failure or have died due to secondary pathogens.

The sampling of treated animals late in the disease (the exception is chronic disease) can yield false results. In chronic disease, pathogens may be contained to specific areas i.e. a granuloma and can be present in very low numbers, so hard to detect.

Selection of the correct sampling site

Generally, sample the affected tissues or organs.

Sample site considerations:

1. Sample the most recent lesions. Chronic lesions can become secondarily infected or show evidence of healing i.e. fibrosis or scar tissue.
2. Sample the edge of large lesions. The centres of large lesions can start to heal or become necrotic and could also contain non-viable pathogens. The outer edge in expanding lesions is the site of recent cell disruptions and where active micro-organisms are present. It also allows the pathologist to determine the edges of tumours and compare normal and diseased cells.
3. Sample several lesions, if present. It is likely that the lesions are in different stages of the disease process and that will show an overall pattern improving diagnostic specificity.
4. Sample as deep as possible when dealing with skin lesions. Superficial sampling can fail to yield the causative agent, in fact, commensal bacteria on the skin may be incorrectly identified as the cause. Superficial sampling of deep lesions can introduce surface contaminants into the sample.
5. Sample using your knowledge of the pathogenesis of the suspected diseases. Exception: you may have to sample differently to the disease site for some diseases. For example, botulism is a bacterial intoxication and the best site to sample to determine the cause is the plasma of the affected animals, not the motor end plates in peripheral muscles where the disease occurs.
6. Sample more accessible sites that are still representative of the original site which would require a more invasive sample. For example, sample urine to detect pathogens of the renal tubules in the case of leptospirosis. However, when possible the original site of the lesions is still a better, but not always practical sample.

Selection of the correct sample type

The type of sample used depends on the sample site, lesion type and the required tests.

A few useful sample types for bacterial and fungal identification are shown in the table below:

Sampling sites and the most useful sampling methods for bacterial and fungal identification

Sample site	Sample type
Urinary tract	Best is cystocentesis (aspirate) in dogs and cats, alternatively by mid-stream urine or if necessary urinary catheter
Endometritis/pyometra	Transcervical swab sing a vaginal speculum or uterine lavage
Prostatitis	FNA of any cysts, urinary catheter to level of prostrate and massage the gland to obtain fluid. Urine can be used
Orchitis	Semen
Diarrhoea	Fresh stool or rectal swab – culture for specific pathogens; PCR swab for known pathogens
Conjunctivitis & keratitis	Bacterial swab for bacteria and fungi; PCR swab for mycoplasmas and viruses
Rhinitis	Biopsies or deep nasal flush for fungal and bacterial cultures; PCR swabs for Mycoplasmas and viruses
Tracheobronchitis & pneumonia	Transtracheal wash or bronchoalveolar lavage
Septic arthritis & osteomyelitis, periodontal disease	Aspirate of synovial fluid, infected implants, surgical biopsy of necrotic bone. Must also culture for obligate anaerobes
Skin	Tissue biopsy from skin pre-disinfected with 70% alcohol that is allowed to dry. Superficial samples removed surface crusts and use a bacteriological swab. Aspirates of pustules and abscesses
Blood	Endocarditis or fever of unknown origin. Up to 10 ml whole blood in a blood culture bottle different venous sites 30 to 60 minutes apart

A special case: What to do when there is a fever of unknown origin (No need to learn – just indicates that a sample like this must be collected aseptically and properly)

You can collect blood for a blood culture from an animal with a fever of unknown origin, bacterial pneumonia, with suspect bacterial sepsis or endocarditis.

1. You should collect 2 to 3 lots of blood samples hourly for acute sepsis and every 30 minutes for endocarditis.
2. Make sure you collect the blood before you initiate antibiotic therapy.
3. You will have a blood culture bottle into which you will collect blood that has been aseptically collected.
4. First, prepare the skin over the vein you wish to collect blood from. In large animals like horses, cattle, sheep and goats you will use the external jugular vein. In dogs and cats, the cephalic veins are usually more convenient.
5. Wash hands thoroughly and don gloves.
6. Shave the skin, locate the vein and block the jugular vein with digital compression and the cephalic vein with a tourniquet.
7. Disinfect the shaved skin with an isopropanol swab, allow to dry and follow with chlorhexidine or povidone iodine and insert a needle, usually 21G that is attached to a 10mL syringe.
8. When possible collect 10 ml of blood. You can collect less for very small animals.
9. Release the tourniquet or finger pressure and then remove the needle.
10. Blood collected through an indwelling catheter may be contaminated, so a slightly different procedure is used. In this case, inject normal sterile saline, then aspirate 6 ml of blood and push it back into the bloodstream. Repeat this procedure twice. With a clean syringe collect 10 ml of blood.
11. Remove the seal from the blood culture bottle, wipe with an alcohol swab and allow to dry.
12. Inject all the blood into it.
13. Gently swirl it to mix the blood with the culture broth in the bottle.
14. Make sure the bottle is accurately labelled.
15. Send the bottle at room temperature to the laboratory within 12 hours of collection.
16. If you don’t have a blood culture bottle, you can directly submit 10 ml of blood in an anticoagulant like heparin to the laboratory. This should reach the laboratory within an hour.

 

Two examples of samples are shown below:

Learning Objective 3

Explain how the use of cytology of the lesions can assist you in deciding whether to submit specimens to a microbiology laboratory.

Cytology

Cytology is a rapid in-practice test that can assist you in determining whether lesions are associated with inflammation or tumours. They are very useful in determining whether a bacterium, fungus, ectoparasite, protozoa or helminth is present. In acute and life-threatening infections,  they can assist you in choosing empirical antimicrobial treatment.

The site you need to sample determines the method used. Below is a table that provides some tips on what sample should be used for different tissues.

Table illustrating the different samples that can be collected for cytology. You will learn how to collect various samples in clinical pathology and medicine.

Sample type	Sampling site/s	Sample processing
Fine needle apirate/insertion biopsy	Solid tissues i.e. nodules, plaques, tumours, lymph nodes, organs, small delicate tissue insertion only no aspirate   Encased fluids: joints, bladder, abscesses, cerebral spinal fluid	Blow the material onto a glass slide and spread with another glass slide   Deposit a drop and spread along the glass slide with another glass slide
Impression smear	Open, exudative or greasy lesions; biopsies or extirpated tissue, skin surface after crusts have been gently removed	Firmly press glass slide to lesion or firmly press the tissue to a glass slide. Moist tissues may have to be dabbed onto a paper towel first
Scrapings	Superficial skin lesions	Smear on the glass slide
Swabs	Fistulas, ear canals, skin folds, holes after punch biopsies eye conjunctiva & cornea	Roll swab on glass slide
Adhesive tape	Superficial skin crusts, seborrhea	Mount directly to slide. Can use a vital stain like lactophenol cotton blue. Wipe off excess fluid
Lavage	Middle ear, respiratory tract, seborrhea	Sterile saline or PBS is inserted down the tube or into the cavity allowed to mix and collected with a syringe attached to a suction tube

When a lot of fluid has been collected, that is urine and lavages, the fluid can be centrifuged, the supernatant discarded and a smear of the sediment made.

The most common stain used in practices is Diff-Quik® or a similar Romanosky-type stain.  Bacteria stain purple. Generally, rods represent gram-negative bacteria and cocci gram-positive bacteria. This is true for urine and most ear swabs. However, there are exceptions. When necessary stain a second smear with the Gram staining method. These stains must be regularly replaced otherwise they will stain poorly or can become contaminated and yield false positive results for the presence of bacteria or fungi.

Examine the prepared slide using a compound light microscope. You will usually start at low power, choose a field of interest and gradually move up in objective magnification to better observe detail. Cells and large infectious agents are best observed up to 400x magnification whereas the very small bacteria are best observed using an oil objective at x1000 magnification. If you are unsure of how to use a microscope click on this link to NCBioNetwork virtual microscope 3D tutorial.

Slide interpretation specifically for bacterial or fungal infections

Indicators of bacterial/fungal infection:

• presence of large numbers of especially neutrophils in acute inflammation and macrophages in chronic inflammation
• bacteria or fungi present intracellularly, especially in neutrophils and macrophages
• both intracellular and extracellular bacteria or fungi
• extracellular bacteria or fungi in the presence of neutrophils and macrophages.

False negative results:

This occurs when bacterial or fungal numbers are low and incorrect sampling. For example, a superficial swab of an eye cornea was collected for fungal identification, but not observed cytologically, as the fungus was present in the deep stroma of the cornea, out of reach from the sample.

False positive results:

• Cocci can be especially confusing as they can be confused with stain deposits, melanin granules, degenerating nuclei and even ingested debris in phagocytes. Look for bacterial behaviour of cocci i.e. cocci in chains or in clumps. Melanin granules tend to have a brown colour that can be observed when gently focusing up and down with the objective.
• Bacterial rods can be confused with stain crystals, fibrin and cell rolls. Always look for an unstained zone around the bacteria as this often represents the bacterial outer membrane and capsule.
• Yeasts can be confused with stain deposits, fat globules, pollen and toxic neutrophil nuclei.
• Fungal hyphae can be confused with cell rolls, fibrin and cotton swab debris.
• Bacterial or fungal contamination of the dipping type stains such as DiffQuik If this happens you will note that the bacterium or fungus is distributed throughout the stain and that you will have a run of slides with the same bacteria or fungus present.

Below are some examples of what you may encounter when examining slides:

Sampling for bacterial or fungal isolation and antimicrobial susceptibility

It is critical that samples collected for bacterial or fungal isolation and antimicrobial susceptibility testing have viable organisms present. Thus disinfectants, antiseptics and even antibiotics should be avoided when collecting these samples and have no chemical preservatives – there are exceptions. It is also important that tissues that are sterile in healthy animals are preferably sampled. The only surface disinfectant that can be used to surface disinfect a sample is 70 to 80% alcohol which is allowed to evaporate before collecting the sample. This is because all other disinfectants have residual activity which will kill the microbes in the sample. No such restrictions are placed on samples collected for serology, PCR or histological diagnosis.

Indications:

1. Patient is not responding to treatment
2. Secondary infections associated with an underlying disease i.e. diabetes mellitus
3. History of relapse or re-infection
4. Presence of rods on cytology = more likely to have antibiotic-resistant bacteria
5. Immunocompromised patient
6. Infection is life-threatening
7. Evaluation of efficacy of treatment during a long course of therapy.

Contraindications 

1. Sampling risk outweighs the benefits. When invasive sampling may exacerbate the current condition i.e. an animal with a clotting disorder may bleed; High-risk patients may not tolerate anaesthesia; a bronchoalveolar lavage may result in contamination of the lower airways in immunocompromised patients. For these patients, you may decide to use a less invasive sample that may be contaminated with commensal microfloraand request that the laboratory test for specific pathogens.
2. Commensal microflora present in the samples hampers the diagnosis e.g. mouth, faeces and external genitalia. Usually, specific pathogens should be sought for conditions involving sites with normal microflora i.e. Salmonella from an animal with diarrhoea.
3. Infections that are treated topically.
4. When patients are responding well to antibiotic therapy

SPECIMEN PRESERVATION, PACKAGING and TRANSPORT

How the sample is preserved and packaged is usually dependent on the test type and the distance the practice is from the laboratory.

Cytology: Immediately label, make a number of smears, air-dry – don’t stain. (Stain only the ones you will look at). Air-dried glass microscope slides should be placed in special plastic or cardboard containers. If not available, then wrap them up separately in paper towels to protect them from breakage. Make sure they are not packaged with formalin as the formalin can penetrate packaging and change the appearance of cells.

You may be able to stain a slide yourself and scan it in digitally and then make the digital scanned slide available for the clinical pathologist to interpret. This will save on costs and time.

Histopathology: In a 1:9 ratio 10% buffered formalin in a screw-capped plastic container. If air transport – leave in formalin 1:9 overnight then in cotton swab/pad soaked in formalin but no free fluid. Make sure these specimens are airtight and separated from fresh samples for microbiology as formalin vaporizes easily and is an excellent sterilant.

Specimens intended for histopathology should not be frozen as the cell architecture will be lost when the tissues are defrosted.

Microbiological analysis (Bacterial and Fungal Culture): fresh tissues– sterile; balanced salts; inhibitors; avoid EDTA for viruses – it inhibits cell cultures.

1. Place microbiological swabs in transport medium – usually provided with the swab. This is a non-nutritive semi-solid medium that contains buffers. To avoid over-growth by contaminants, bacteria should only survive, not grow in this medium. However, some microbes will still grow in it, so keep them cool.
2. Fine needle aspirates are squirted onto a microbiological swab. If the volume collected by the fine needle aspirate is large enough >0.5mL, it can be placed in a sterile plastic specimen container of appropriate size.
3. Tissue biopsies if very small (< 3mm wide) can be placed on a microbiological swab in a bacterial transport medium. If large, they should be wrapped in sterile saline-moistened surgical swab and placed in a sterile, plastic specimen (screw cap) container of appropriate size.
4. Specimens for anaerobic culture should either be in a transport medium that keeps the REDOX low or be placed in an anaerobic or carbon dioxide-enriched environment i.e. GasPaks. Carbon dioxide gas is heavy and will replace oxygen around the specimen. Simple carbon dioxide generators include dry ice and the use of bicarbonate antacids in a small amount of fluid. Another solution is to ensure that the size of the specimen is > 5cm in diameter and fills the entire container. The centre of the specimen will remain anaerobic. The transport medium for swabs can also keep small specimens anaerobic for a short time, so send those to the laboratory ASAP.
5. Urine samples should be kept refrigerated and reach the laboratory within 24 hours. For longer transport buffered boric acid can be used.

PCR: dry swabs; filter paper; blood in EDTA; fresh tissue biopsies. These specimens can be stored at room temperature if in an RNA/DNA preservative or on filter paper. Samples that are not dry should be either cooled or frozen. This is to stop the growth of contaminants that may produce DNAses or RNAses that degrade the target genetic material. The large water crystals that form in cells during freezing actually assist in the extraction of DNA or RNA from cells. Although DNA is very stable, RNA degrades very rapidly in specimens.  Some companies provide special in DNA/RNA preservative i.e. RNAEASY.

GENERAL SPECIMEN PACKAGING

1. Place all specimens in a leak-proof, non-breakable container that is properly labelled. Screw-capped containers are best.
2. Ensure that the specimens have no contact with light as UV light is destructive to microbes and will destroy DNA.
3. Ensure that packaging can mop up any spills or contain any breakages
4. Infectious agents require special packaging. Category A agent samples:  Label UN 2814 is for agents that can infect people and animals; and Label UN2900 for agents that only infect animals.  Category A agents consist of cultures of pathogen risk groups 2 (some), 3 and 4 and samples that are suspected to have risk group 4 pathogens i.e. Hendravirus. Standard diagnostic specimens that are suspected that have risk groups 2 and 3 in them are in Category B. They get a UN3373 label. The picture below shows the packaging required for Category A and B agents. Category C are samples that are excluded from the regulations. These include biological and environmental samples that are not suspected to be infected and those samples where the infectious agents have been inactivated i.e. formalin-fixed specimens and dried specimens for PCR.
5. A link to an excellent YouTube video on how Category A and Category B samples should be packaged and labelled. It has been developed for veterinarians and paraprofessionals by Queensland Biosecurity Services Laboratory.

SPECIMEN SUBMISSION FORM & SPECIMEN TRANSPORT

1. Please fill in the form as completely as possible. The laboratory will know what tests you want; be able to suggest other tests as appropriate;  and samples won’t be rejected.   Knowing that an animal is on antibiotic therapy may allow the laboratory to use a means of inhibiting the antibiotic to encourage bacterial growth and they can also test whether the bacterium is resistant to that antibiotic.
2. Also ensure that the laboratory can read your writing.
3. The submission form is usually placed separately to the specimen to protect it from specimen leakage.
4.   Ensure that the required temperature is maintained. Most microbiological samples will transport well at refrigeration or slightly above this temperature, about 10°C. Histopathology and cytology at room temperature 18-25°C – not refrigerated. Dry specimens and specimens in DNA/RNA preservative can also be stored at room temperature. Note that it is often very warm in tropical and outback environments, so the use of a polystyrene or coolbox will ensure that the specimens will never be exposed to these temperatures.
5. Make sure the laboratory you use can do the required test/s
6. Send via a specially registered courier – laboratories often contract couriers to do this. Note that couriers have to be registered to transport Category A and B samples.

Below is a sample submission form used by Pathology Diagnostic Services, James Cook University. Each laboratory will have its own submission form.

Criteria for specimen rejection by the laboratory

1. Mismatch of information on the label and the request
2. Inappropriate transport temperature
3. Excessive delay in transportation
4. Inappropriate transport medium
   • specimen received in a fixative when it should be in no preservatives
   • dry specimen that should have been kept moist
   • specimen with questionable relevance.
5. Insufficient quantity
6. Leakage