Pathology

The VDL Pathology Section offers necropsy services, gross 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.

• Submission Guidelines
• Testing Schedule
• Necropsy Services
• Returning of bodies and cosmetic posting 
• General Guidelines for Field Necropsy
• Preparation of 10% neutral buffered formalin
• Histochemistry and Immunohistochemistry
• Quick Guides to sample selection for diagnosis of common conditions
  • Bovine
  • Porcine
  • Ovine
• Abortion Serology
• Specific Diseases

Testing Schedule

Our goal is to provide accurate results and timely service. However, many diagnostic assays are not performed daily and/or testing schedules may change. If your case involves testing animals for export, testing show animals, testing animals to be sold, and/or testing a large number of samples, please contact the VDL (515-294-1950) prior to submission to verify the availability and turnaround time of specific assays. A phone call will often shorten turnaround time by allowing us time to prepare for testing prior to receipt of samples.

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

As of September 15, 2008, no animal carcasses or ashes will be returned/released to veterinarians or animal owners.  Arrangement for pickup by authorized crematory service is an option.

No cosmetic posts 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.

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).

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').

Tongue

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

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.

Thymus

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

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.

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.

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.

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.

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.

Stomach

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

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.

Current Immunohistochemical Tests

Abortion Serology

Fetal serology 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.

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.
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 (1:2 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 <1:2-1:32, occasionally 1:32-1:128 after boosters.  Recent infection may stimulate antibody titers of 1:64-1:256.  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 1:256 (occasionally over 1:10,000) that persist for months.  Vaccination titers rarely exceed 1:256 and begin to drop by 2 months.  A titer of 1:512 or greater 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.

Pseudorabies virus (PRV)

In an endemically infected swine herd, PRV neutralizing (VN) antibody titers will range from 0 to 1:256.  Recently infected pigs will usually have titers up to 1:32 or greater.  Vaccination titers rarely exceed 1:16.  Differential tests also distinguish vaccinated pigs from naturally infected pigs. 

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.