Ministry of Agriculture and Lands
BC Ministry of Agriculture and Food
Animal Health Centre
1767 Angus Campbell Road
Abbotsford BC V3G 2M3
SPECIMEN SHIPPING INSTRUCTIONS
We reserve the right to discard unsatisfactory specimens
General
Specimen Submission for Abortion Diagnosis
Disease Investigation for Poultry
Specimen Submission for Virus Diagnosis
Specimen Submission for Histopathology
GENERAL
1) Direct delivery - 8:30-12:00; 1:00-4:30,
Monday - Friday
Please deliver as early in the day as possible.
2) Post - Samples must be adequately packed so they do not leak*. They must be well preserved so that they arrive in suitable condition for examination.
3) Bus - samples must be adequately packed so they do not leak*. Collected from Abbotsford Bus Depot by laboratory staff throughout the day.
4) Courier. Numerous companies deliver specimens directly to the AHC.
History
A complete history indicating the disease(s) suspected is
essential for all submissions, including tumors and biopsies.
Please enclose submission information in a separate plastic bag
and pack it at top of box.
Tests
Make sure that the correct test is noted on the submission form or samples will be placed on hold for 2 weeks then discarded.
Diagnosis
Is based on the history provided and the results from the various
sections in the laboratory. The Laboratory results must be
coupled with conditions on the farm, such as management,
nutrition, environmental conditions, etc. For best utilization of
our service a complete history and good, well-preserved specimens
should be submitted.
Preservation
a) Refrigeration: Use a styrofoam box, frozen picnic packs or
balloons partly filled with water and frozen, and insulate well
with newspaper, sawdust, etc. Protect and separate samples in
leak-proof plastic bags.
b) Fresh Frozen: Tissue in a sterile container, frozen and shipped with ice packs.
c) Formalized: When requesting histopathology, submit tissues in formalin rather than fresh tissues. This will prevent autolysis while in transit.
Send several pieces of tissue no thicker than 3-5 mm; length and breadth can be several centimeters.
One part tissue to 10 times volume of 10% neutral buffered formalin (see attached recipe or consult AHC for buffering agents).
* Specimens shipped frozen are often thawed by the time of arrival, and unless packed well, will leak.
NOTE: Please do not freeze samples for which you may require histopathology.
SPECIMEN SUBMISSION FOR ABORTION DIAGNOSIS
Abortion Submissions and Specimens
The history of the dam should indicate whether primiparous or multi-parous, nutritional status, previous illnesses or stresses, vaccination history, general level of management and herd health status, recent additions to herd, previous abortions or indications of infertility.
Very often specific abortifacients may only produce lesions or characteristic changes in one or a few tissues. To maximize the possibility of detection of these agents, submit all fetal tissues, including placenta, heart blood and stomach contents on a routine basis.
With swine, sheep, and goats it is usually convenient to submit entire fetuses. The specimens should be chilled (not frozen) and packed with sufficient ice to keep them chilled until they arrive at the laboratory. Bacterial decomposition generally is not the problem with aborted fetuses that it is with carcasses of animals born alive. (1) With modest care, abortion specimens will arrive at the laboratory in condition suitable for examination.
Because of their size, it is often more convenient for the practitioner to necropsy aborted foals and calves and submit selected tissues to the diagnostic laboratory.
The following specimens constitute the minimum requirements for a complete examination:
Frozen or refrigerated:
1) Placenta: (if available). Very important. Entire placenta from horses, sheep and swine if possible: two or more cotyledons, especially with any lesions, from cattle.
2) Stomach contents: A few ml collected in a syringe.
Send chilled.
Fetal heart blood: A few ml chilled.
3) Dam's serum: Draw 5 to 10 ml of blood and allow it to clot overnight at room temperature. Remove the clot the following day and submit serum with other specimens. Preferably follow with a convalescent sample in two weeks.
4) Fetal tissues: Formalin-fixed in 10% neutral buffered solution:
- eyelid (both skin and conjunctival surfaces)
- thyroid
- lung
- spleen
- adrenal
- heart (portion of both ventricles)
- ileum with portion of Peyer's patches
- thymus
- liver
- kidney
- brain (portions of cerebellum, brain stem, and cerebrum)
Fresh chilled fetal tissues: lung, liver, kidney, spleen. Fetal heart blood.
Supplementary tissues or submissions:
Urine: Useful in leptospiral FA tests. Send chilled, with one drop of formalin per 20-30 ml urine.
Milk: May be useful in leptospiral tests. Fresh and preserved as with urine.
Amnion: Useful in Ureaplasma diversum diagnosis. Send formalin fixed and fresh chilled, along with other routine samples. For storage, -70C is preferred to -20C. Send chilled samples promptly.
Brain: Entire brain, formalin-fixed. Useful in toxoplasma abortion in sheep, goats. Useful in BVD abortion and Neospora in cattle.
For Vibrionic abortion (Campylobacter fetus ss fetus): tampons and containers bags must be provided by this laboratory. Avoid fecal contamination. Refrigerate or freeze.
** If there are any questions phone and speak to one of the pathologists.
Reference:
1) Kirkbride, CA. Laboratory Diagnosis of Livestock Abortion, 3rd
edition, 1990. Iowa State University Press, Ames, Iowa.
DISEASE INVESTIGATION FOR POULTRY
Modern agricultural economic practices dictate that a large number of birds be raised in close proximity, in confinement housing and within an artificially controlled environment. This, along with genetic selection for break-neck productivity imposes a predominant physiological stress which can manifest itself in a variety of ways, most importantly by increased susceptibility to infections and production related disease. The expression of ill health can be tempered or exacerbated by management level and dedication of the farm manager. In the course of disease investigation, the poultry practitioner must cover a lot of ground since the majority of production problems are directly related to intensive management systems rather than infectious disease.
The first step is to clearly define the problem. As always, a complete and relevant history is critical. It should include flock health program information such as the vaccination schedule (timing, type, mode and any noted reactions), baseline ELISA serology profiles and previous treatment protocols. Current performance should be compared with known standards of production or the production records of previous flocks. On farm record-keeping is vital since it allows early detection of problems and also makes obvious to the producers the benefits of your recommendations for treatment or management changes.
Flock problems present themselves as increased morbidity, mortality or production drops. "Acceptable" levels of mortality for each type of production bird exists within the industry. For example, 4% mortality is acceptable at the end of 6 weeks in broiler chickens and one half of 1% per month during the lay cycle is acceptable in laying birds. We tend to get more excited about death losses but we must consider that most financial losses are due to reduced productivity and subclinical disease rather than mortality.
Problem definition should also include the number of birds affected, the clinical signs, if any, and the onset and duration of the problem. Can these be correlated to recent changes in personnel, feed load or weather? On-farm investigation involves systematic evaluation of management and can be facilitated by using the FLAWS guideline. FLAWS stands for:
| Feed: | (consumption, efficiency, quality) |
| Litter: | (type, quality, moisture) |
| Air: | (ventilation, temperature, dust, NH3) |
| Water: | (spacing, quality, sanitation) |
| Space: | (density: 0.75m2 per broiler, 3 vs. 4 layers/cage) |
Now look at the birds. Can coughing or snicking be heard or is there any change in activity? Are birds distributed evenly throughout the barn? Is there size uniformity? If the problem is morbidity or mortality, examine sick or dead birds. If the problem is poor growth, focus on the smaller birds. If there has been a drop in egg production, examine birds with visible signs of going out of lay (pale, withered combs, less than 3 fingers between pin-bones, feather molting, etc.). The advantage of working with flocks in the tens of thousands is that a small sample of "representative" birds can be sacrificed to assist in determination of a diagnosis. The Animal Health Centre can assist you with laboratory back-up.
Commercial poultry works on a low margin cost of production and a tightly defined production schedule. If a disease problem persists or has high mortality losses then both the cost breakdown and production cycle are thrown out of line for the rest of the life of that flock. This loss is never recoverable and for this reason poultry producers, more than any other type of livestock producer, require and demand immediate diagnosis and action.
While some diagnostic procedures take time, poultry practitioners must provide tentative diagnoses based on history, on-farm inspections and gross postmortem findings and make immediate action recommendations which can later be modified as new information becomes available.
SUBMISSION OF SPECIMENS FOR VIRUS DIAGNOSIS
1) All isolation procedures for virus agents are delicate, difficult and time consuming. Only tissues from freshly dead animals are satisfactory for virologic examination. Autolyzed tissues are useless.
2) Tissue Specimens for virus isolation should be collected promptly, placed in an appropriate container (plastic bags are excellent) and kept refrigerated or frozen as soon as possible. The use of ice pack refrigerants to keep the specimens cold while in transit is extremely important for for virus diagnosis. If specimens are frozen, they must remain frozen in transit and not be allowed to thaw out. If you are not sure that specimens will remain frozen, please send sample refrigerated only.
3) Individual viral culturettes (used for nasal, tracheal, anal and cloacal swabs) are a sterile, disposable collection and transport system for viral specimens only. They cannot be used for bacterial culture, likewise bacterial culturettes cannot be used for virus isolation. Virus culturettes containing media/antibiotics are preferred because they allow direct electron microscopy (EM) as well as isolation (cultivation) procedures. Culturettes containing gelatin or agar are unsuitable for direct EM.
4) Viruses can be denatured by light, heat, dehydration and pH changes among other things. Therefore specimens and swabs must be sealed.
5) All avian serology except chick anemia agent (CAA virus) will be performed by the Animal Health Monitoring Laboratory (AHML). Information and charges concerning avian serology can be found the AHML section.
6) Virus isolation and identification procedures can take from one day to several weeks, depending on virus type and procedures required. Call the Animal Health Centre for information on length of time for identification or for any other information.
HISTOPATHOLOGY
Whenever possible, fix tissues at the time of necropsy if histopathology is required. Transit time invariably results in tissue autolysis if specimens are sent in fresh; better to fix them prior to submission.
Freezing seriously impairs histopathological examination of specimens by causing extensive disruption of cellular structures. Always send fixed tissues rather than frozen, if histopathology is required.
Preparation of fixed tissue with formalin:
Formalin will only penetrate approx. 3 mm in 24 hours, so specimens should be no thicker than 5 mm. Other dimensions are not critical but should be large enough to provide an adequate field of study. Ten volumes of buffered fixative to one volume of tissue is essential for adequate fixation.
If larger pieces of tissue, such as brain, are to be sent it is best to make some cuts so that formalin can penetrate more quickly. Once fixed, transportation to the Animal Health Centre using just enough formalin to cover the fixed tissue is adequate.
Select a section of tissue at the edge of the lesion to include some normal as well as diseased tissue.
We have included a recipe for 10% neutral buffered formalin. If this formulation is followed carefully, your fixed tissue preparations will be satisfactory. If formalin is not buffered, acid hematin (a blackish-brown pigment) forms from hemoglobin and can impair microscopic examination and obscure subtle tissue alterations.
The Animal Health Centre will be pleased to provide these buffering agents in a quantity sufficient to make up four litres of 10% neutral buffered formalin.
Recipe for 10% Neutral Buffered Formalin
Formalin 35-40% strength - 10 ml
(Na H2 PO4 H2O) Sodium phosphate
monobasic monohydrate - 0.4 gm
(Na2 H PO4) Sodium phosphate dibasic
anhydrous - 0.65 gm
Water - to 100 ml
