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Pathology

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The VDL Pathology Section offers necropsy services, gross examination and histopathological evaluation (histochemistry and immunohistochemistry) of tissue samples. Telephone consultation with a VDL pathologist prior to the submission may be useful and is particularly encouraged if a case is complex or the diagnostic investigation is an ongoing series of submissions. Please call the laboratory (515-294-1950) and talk to one of the diagnostic pathologists at any time if you have questions concerning specimen selection or preservation.

Pathology Submission Guidelines

Submission of tissue specimen(s) is usually the best method of obtaining a diagnosis of a clinical disease. However, proper selection and preservation of samples is essential to make the most efficient and economical use of the laboratory. The two conditions that most frequently interfere with diagnosis are (1) post mortem autolysis, and (2) sample collection too late in the course of disease.

Fresh specimens should be large enough to demonstrate the lesion yet small enough to allow for rapid chilling (tennis-ball sized). In some cases (e.g., bronchoalveolar lavage for PRRS virus isolation) submission of an entire organ may be preferred. Ideally, fresh samples should be packaged individually to prevent cross-contamination. Please do NOT to package fresh intestine with other tissues, as this results in fecal contamination of other organs. At a minimum, intestinal samples should be submitted separate from all other tissues. Samples should be shipped in leak-proof containers with artificial freeze packs. Please line shipping containers with additional sealed plastic bags to avoid leakage in transit.  Carriers will not deliver leaky packages resulting in delays in processing.  A fee of $25 may be assessed for packages that leak.

Specimens for histopathology should include thin (0.5-1.0 cm or 1/2 inch) slices of the appropriate organs, including the lesion, transitional zones, and adjacent grossly normal tissue, in 10% buffered formalin in a leak-proof, wide-mouthed solid container. Do not use glass containers. When in doubt, collect specimens from multiple organs, including brain. The pathologist can then select the most appropriate specimens for complete microscopic examination. Unless it is important that individual animals be examined and reported separately, specimens from each individual animal can be pooled in a single container - provided the specimens are small enough to maintain a 10:1 formalin:tissue ratio. The ratio of formalin:tissue should be at least 10:1 to allow optimal fixation.

A minimum of 48 hours is required for histopathology, 24 hours for fixation and 24 hours for processing. Consequently, specimens for histopathological examination should be collected and placed in formalin at the time of necropsy in order to minimize autolysis and generally allow results to be available the day after the specimen is received.

General guidelines for histopathologic examination/submission of formalin-fixed specimens are as follows:

  1. Solid organs: 0.5 - 1 cm slices to include lesion and transitional areas between lesions and normal tissue.
  2. Intestine: 1 - 2 cm lengths held open as they are immersed in formalin or flushed with formalin prior to immersion. Do NOT tie the ends.
  3. Brain (including brain stem): immerse sagittal half of small brain in formalin. One-half of the brain should also be submitted from larger animals. These larger brains should be sliced for better fixation. The preferred procedure is to make transverse slices, 1.0 - 1.5 cm apart that extend to within 1.0 cm of the ventrum of the brain. This allows for more rapid fixation while retaining the architecture of the brain so that specific areas can be sampled. Nerves can be pinned to a tongue depressor and immersed in formalin.
  4. Tumors: immerse in 10% formalin. If greater than 1 cm, incise, leaving a base attachment intact.
  5. Impression smears and needle aspirates of tumors are helpful in a few situations. However, actual biopsies are usually required for definitive diagnosis. Stain and examine smears and aspirates in your practice laboratory and, if a diagnosis is not obvious (i.e., mastocytoma, abscess, etc.), biopsy the mass and forward both the biopsy and the smears to the reference laboratory.

Necropsy Services

Veterinary pathologists necropsy carcasses, evaluate fresh tissues grossly, and evaluate formalin-fixed tissues microscopically for the presence of lesions. Live animals and intact carcasses may be submitted to the laboratory for examination. Live, untreated animals with signs typical of the acute disease, or dead animals without postmortem decomposition are the submissions of choice. Tests will NOT be performed on specimens deemed unsuitable (autolyzed or incorrect samples). A complete history should be included with all submissions. Special procedures or circumstances (legal issues or insurance cases) should be designated as such at the time of submission.

Returning of Bodies and Cosmetic Posting

No animal carcasses, body parts or ashes will be returned/released to veterinarians or animal owners.  Arrangement for pickup by authorized crematory service is an option.

No cosmetic necropsies will be performed on animals/bodies submitted to the laboratory for necropsy/testing.

Please call the Laboratory Director with questions and additional details relating to these policies.

General Guidelines for Field Necropsy

When collecting tissues from necropsied animals, the history, clinical signs, and gross lesions should determine which tissues are collected. The following sampling recommendations and reminders may be helpful in optimizing the diagnostic information that can be obtained.

Brain

Sever the cranial cervical spinal cord as far caudally as possible when removing the head. Sagittally section brain and submit one-half in formalin and one-half chilled. Be sure to include cerebellum and brainstem in addition to cerebrum. Brain should always be submitted from animals found dead without gross lesions in the thoracic or abdominal viscera.

Gross lesions

ALWAYS submit representative samples of ANY gross lesions. For example, oral erosions/ulcers are often described on submission forms but less often submitted, especially in suspect BVD-mucosal disease cases. Fixed ulcers are an excellent sample for detecting BVD virus by immunohistochemistry (IHC).

Heart

Open all chambers and examine all valves. If lesions are identified, submit formaliin-fixed and fresh (if infectious agents are suspected) samples. If no lesions or evidence of heart failure are identified, submit a fixed portion of papillary muscle from the left ventricle.

Intestines

When sampling the intestinal tract, always begin at the ileocecal junction and work toward the stomach. Examination of multiple areas is often required, since lesions and/or agents are usually not uniformly distributed throughout the intestinal tract. Place 3 widely spaced 1-2 cm segments of ileum, 3 segments of jejunum, and one segment of duodenum in formalin, making sure that the formalin contacts the mucosal surface. Cecal contents should be collected for virology, and a portion of cecum should be fixed. Samples of several areas of spiral colon should be placed in formalin so that both ascending and descending loops will be included. As noted above, also submit any gross lesions. If the animal is of small size, the entire remaining intestinal tract may be submitted chilled. Otherwise, representative loops of ileum, jejunum, and colon should be submitted. Also, include fixed and fresh enlarged lymph nodes.

Joints

Joint fluids need to be sampled using aseptic techniques. Synovium should be submitted on ice and in formalin. Submission of intact joints on ice is an alternative.

Liver, kidney, spleen

Since these organs have many potential uses for the diagnosis of many different infectious agents and diseases, please submit formalin-fixed and fresh samples in all cases.

Lung

Submit both formalin-fixed and fresh tissue samples from at least four different areas: cranial, hilar, middle, and caudal lung. More extensive sampling greatly increases the probability of diagnosis. Fresh portions of lung should be as large as possible. The entire lung (less the parts collected for formalin fixation) should be submitted, if possible. If lung lavage for PRRS virus isolation is desired, submit one entire (unsampled) lung. Also, include both fixed and fresh enlarged lymph nodes.

Lymph nodes

Lymph nodes, especially those enlarged or hemorrhagic, are useful for detection of viral infections, bacterial infections or neoplasms. Please include fresh and formalin-fixed lymph nodes (and tonsil) when appropriate. 

Skin

Skin lesions should be submitted fixed and fresh. Nonlesional skin from dead bovines is useful for determining BVD virus persistent infection status. Ear (pinna) or coronary band are preferred sites (see discussion under 'Special Tests').

Spinal Cord

When clinical signs suggest spinal cord involvement, it is essential to submit at least parts of spinal cord. A Barnes dehorning tool works well to gouge away bone to access segments of cord. A cleaver, ax, hatchet or saw is also effective. Segments representing cervical, thoracic and lumbar regions in formalin and on ice are appropriate specimens. Alternatively, submit vertebral column or vertebrae on ice.

Stomach

Submit any lesions in formalin. In ruminants, submit one piece of fixed abomasum even if no gross lesions are present.

Thymus

Fixed and fresh thymus are useful for detecting BVD virus in cattle and PRRS virus in pigs.

Tongue

Formalin-fixed tongue is useful for detecting generalized myopathies, especially in fetuses (Neosporacaninum) and neonates. Muscular diaphragm is also useful for this purpose.

Trachea

In general, formalin-fixed and fresh trachea is only useful if gross lesions are present. In cattle, hemorrhages are common but nonspecific in animals with respiratory disease. Pooled exudate from lung is easily wiped away to reveal a smooth, glistening (normal) mucosal surface. This feature distinguishes pooled exudate from an adherent fibrinonecrotic membrane, such as is typical of IBR virus.

Preparation of 10% neutral buffered formalin

Buffered formalin concentrates are available from several suppliers. These are ready for use after the addition of water. Non-buffered 10% formalin (1:9 of 37% formaldehyde:water) can be substituted for neutral buffered formalin in emergency situations. Do NOT submit samples in undiluted 37% formaldehyde.

10% neutral buffered formalin is prepared as follows:

  • 900 ml distilled water
  • 100 ml 37% formaldehyde solution
  • 8.0 g NaCl
  • 4.0 g potassium phosphate monobasic
  • 6.5 g potassium phosphate dibasic

Histochemistry and Immunohistochemistry

Various histochemical techniques are available to demonstrate agents/structures within histologic sections. These include the following:

Histochemical staining

Histochemistry demonstrates bacteria, fungi, cellular components, etc., by use of differential stains, e.g., a Gram stain for demonstrating bacteria or Giemsa stain for mast cell granules. 

Immunohistochemistry (IHC)

IHC is used to demonstrate specific antigens within tissue sections. The location of agents (bacteria, viruses) can be visualized within the tissue in association with lesions. This technique is performed on formalin-fixed specimens. Results are generally available 24 - 48 hours after receipt, providing the proper specimens are submitted in formalin.

See the VDL Fee Schedule for a current listing of immunohistochemical tests. 

Abortion Serology

Fetal serology from fetal thoracic fluids has not proven to be of much value in abortion diagnosis.  In sheep, the diagnosis of toxoplasma abortion can be accomplished by detection of antibody in fetal fluids.  Fetuses are rarely capable of producing antibody until the last trimester.  Fetal thoracic fluids are useful for detection of viruses and should be included in submissions.

Serological analysis of maternal serum can aid in the diagnosis of abortion under limited circumstances with some diseases.  The lack of antibody can be considered evidence ruling out a given disease.  It is more difficult to establish guidelines that point toward a diagnosis. 

Additional points to consider would include the following:

  1. Paired serum samples (at the time of abortion and 2-3 weeks later) may provide more information (i.e., a 4 fold increase in titer) than a single sample.  However, as a general rule, the dam has seroconverted at the time of abortion and titers are often maximal or declining.
  2. As an alternative to paired samples, comparing mean antibody titers and percent seropositivity between affected and unaffected groups of animals may be helpful.  Samples should include serum from approximately 20 aborting animals and at least an equal number of matched (as closely as possible by age and stage of gestation) unaffected animals.
  3. With some diseases, many herds are endemically infected and a majority of the animals will have titers.  Examples:  parvovirus in swine, toxoplasma in sheep, BVD in cattle.  Presence of antibody against these organisms in aborting animals is not sufficient evidence of cause.  However, high titers may indicate recent infection.  The absence of antibody titer could rule out several agents as causal.
  4. Response to vaccination also can complicate interpretation of titers.

Specific Diseases

BVD virus

Most cattle herds are infected with BVD virus.  Virus can circulate within a herd without observable problems.  In such herds, many animals will have (SN) titers (2 to >4,096).  Vaccination titers vary considerably with the type (killed vs live) vaccine used.  Individual animal tests usually are of little value.  Paired samples on a portion of the herd may provide information on virus activity within the herd.  Antibody titers to both type I and type II BVD viruses should be determined and evaluated in light of the herd vaccination history.  Antibody titers in precolostral neonates and unvaccinated calves over 6 months of age are almost always significant.

IBR virus  

Vaccination (SN) antibody titers in cattle usually range from <2-32, occasionally 32-128 after boosters.  Recent infection may stimulate antibody titers of 64-256.  Paired samples on a number of animals to determine virus activity within the herd may be of more benefit than single samples.   Aborted fetuses are usually autolyzed in utero, so fetal serology is not generally useful.

Leptospirosis  

In cattle, infection with serovars other than hardjo usually will stimulate (MAT) antibody titers of 1:800 or greater that will stay high for a few months.  Cows aborting due to leptospirosis may be ill, though illness may not be obvious, especially with serovar hardjo.  Vaccination antibody titers usually will peak at 1:800 for a short time, but higher titers may be seen occasionally in recently vaccinated animals.  Serovar hardjo infection does not stimulate a strong antibody response.  Serology is not reliable in diagnosing infection with this serovar.  An antibody titer of 1:400 would suggest recent infection with this serovar.

In swine, similar comments would apply, but aborting sows and gilts usually will not be ill.   Even higher antibody titers (1:32,000) may be seen occasionally in infected swine.  In this species, serovar bratislava is the host-adapted strain to which minimal antibody response is expected and antibody titers resulting from infection with serovar bratislava rarely exceed 1:400. 

Parvovirus (PPV)

Nearly all swine herds are infected with PPV.  Natural infection usually stimulates (HI) titers, greater than 256 (occasionally over 10,000) that persist for months.  Vaccination titers rarely exceed 512 and begin to drop by 2 months.  A titer of greater than or equal to 512 suggests exposure to natural infection at some time.

PRRS virus

Results of the commercial ELISA currently used are reported as S/P ratios, not as actual antibody titers.  An S/P ratio of 0.40 or greater is considered positive with strong positives reported in the 1.5-4.0 range.  Recent or active infection may result in value up to 2.5.  Differential tests for distinguishing field infection from vaccine are currently NOT available.

Toxoplasmosis

Not all sheep herds are infected with Toxoplasma gondii.  Antibody titers remain for years after infection, thus the presence of antibody is not evidence for current problems.  A serum antibody titer merely indicates the animal was infected at some time.

Special Tests

Diagnosis of BVD virus persistent infection (PI)

Ear notch biopsy is a method to detect cattle persistently infected with BVD virus.  BVD virus is present in epidermis and hair follicles throughout the skin of PI cattle.  The ear is a convenient site and can be sampled with a small swine ear notcher.  Ear notch specimens (at least 1.0 x 0.5 cm) submitted in 10% neutral buffered formalin are processed for immunohistochemistry (IHC) to detect BVD viral antigen.  This test offers several advantages: 

  1. Testing can be conducted on animals of any age. 
  2. Maternal antibody does not interfere with detection of BVD virus in skin.  
  3. Identifying PI’s as early as possible allows them to be removed from the herd prior to the next breeding season.  
  4. Samples may be collected by producers.
  5. Samples do not require refrigeration.  Samples should be submitted within 7 days after being placed in formalin.  
  6. Six specimens can be processed on one slide; therefore, submissions in multiples of 6 are preferred. 

Please submit specimens in numbered (1, 2, 3, . . . ) snap-cap tubes (not in plastic bags) half-filled with 10% neutral buffered formalin.  Use a VDL Serum Submission form to write the calf ID number next to the tube number, and write ‘BVD virus IHC‘ on the form.  Producers should be instructed to disinfect notchers between calves and to avoid inhalation of and skin contact with formalin.  Questions regarding this test should be addressed to Dr. V.L. Cooper. 

Positive animals may be retested in 10 days to confirm PI status.  They should be isolated until testing is complete.  Evidence indicates that ear notch IHC positive calves are PI, or CI (congenitally infected), both of which can have negative impact on herd health.  

Diagnosis of anaplasmosis using blood smears

Please submit air-dried smears of blood collected in EDTA.  Smears should be made immediately after blood is collected.  Parasites disassociate from RBCs over time and, for that reason, we prefer receiving smears rather than whole blood in EDTA.   If serum is available, serology is probably more reliable than examination of blood smears for diagnosis of anaplasmosis.

Clinical Pathology

Clinical pathology (urinalysis, hematology, serum chemistry, cytology, endocrinology, etc.) is generally not offered through the VDL.  Specimens that only require routine clinical pathological evaluation should be submitted to commercial reference laboratories or arrangements made directly with the Clinical Pathology Laboratory in the Department of Veterinary Pathology (515-294-3282).

QUICK LINKS

 

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