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Tissues should be no thicker than 1 cm. Tissues should be placed immediately in 10% buffered formalin at a ratio of 1 part tissue to 10 parts formalin.

1 Duodenum, two (up to 5 cm long) segments

2 Pancreas (1 cm section, can be left attached to the duodenum)

3 Jejunum (proximal, mid, distal), at least three (up to 5 cm long) segments

4 Ileo‐ceco‐colic junction (these regions can be sampled individually, or this region can be sampled in its entirety. If the latter is chosen, the sample should be opened enough so that formalin can perfuse the mucosa throughout the section, but do not scrape or touch the mucosa while handling.) See Figures 5.6 and 5.7.

5 Colon, distal, one sample (proximal is included in sample listed in d)

6 Liver, up to 1 cm wide sections (from all distinct lobes, including gall bladder)

7 Mesenteric lymph node

8 Any regions perceived to be abnormal

1 Microbiology:Screening for bacterial or fungal organisms of significance can be performed on feces, small intestinal contents, or a combination of both. Be aware that antibiotic therapy can skew or prevent the culture of many bacteria. Any pre‐mortem therapy should be noted in the submission form and on the necropsy report. Feces can be collected in any number of different sterile or clean containers, including bags, urine cups, or tubes. If feces are submitted, it should be specified on the request that significant enteric organisms (such as Salmonella, Clostridia, and Campylobacter) are of concern. Culture results need to be correlated with histologic findings; Clostridia, for example, can be cultured from normal intestines, so histologic correlates or toxin testing has to be performed concurrently with culture. Salmonella, although always significant for herd health and zoonotic reasons, can be shed asymptomatically in cats and dogs. Be aware also that so‐called “commensal” (usually non‐pathogenic) organisms can become virulent (e.g. some strains of E. coli). Diagnosis in these cases would require a combination of histologic and microbiologic results or specialized microbiologic analysis. Specimens destined for culture should be transported and processed as soon as possible; delays of more than 48 hours are undesirable. If processing is delayed, refrigeration is preferable to storage at ambient temperature; freezing will kill many types of bacteria (see adjunct diagnostics).Figure 5.6 The intestines, extending from the gastroesophageal junction (arrowhead) to the distal colon (asterisk) have been removed. In the case of GI disease (or for any complete necropsy) the ileo‐ceco‐colic junction (bracketed by arrows) is one of the important sections for submission.Figure 5.7 The ileo‐ceco‐colic junction is pictured. The intestine can be opened along a sagittal plane for greater penetration of the formalin fixative.

2 Molecular diagnostics:Detection and characterization of pathogenic organisms increasingly rely on DNA or RNA amplification techniques (PCR). These samples need to be taken early in the postmortem from tissue minimally manipulated, but the tissue can be frozen immediately and used at a future date if warranted. DNA or RNA from the infectious agent will degrade at rates dependent on time, environmental factors (temperature, pH) and the organism itself. Ideally, samples from affected organs are fresh or fresh/frozen for molecular analysis. Most diagnostic laboratories or veterinary schools can offer guidance and a list of possible tests, the preferred or potentially useful tissue samples, and the preferred method of shipment. Whole blood (e.g. heart blood) or highly vascularized tissues (spleen, liver, lung) are reliable sources for circulating infectious agents (bacteria, viruses, or hemoparasites).

Individual PCR tests and enteric PCR “panels” are available in many commercial laboratories and veterinary schools. For enteric disease, feces are usually the sample of choice for DNA/RNA retrieval, but individual laboratories vary and should be consulted directly. There are important considerations for whether and when to use PCR for diagnostic analysis. (See Chapter 4 for more details.) For accurate assessment of causation, PCR results should be correlated by histopathologic analysis of formalin‐fixed tissue. For example, if Salmonella spp. is detected by PCR, the correlative lesion of Salmonella associated GI disease is an acute and necrotizing enteritis. It is true that if many cases in a single outbreak are evaluated by a single test, correlation and causation can be established by epidemiologic findings (e.g. only and all cases of enteric disease are positive for Salmonella). However, this can be both unfeasible (depending on the incidence of disease) and expensive. Using histopathology, correlated with PCR (or a number of other diagnostic tests), can establish causation in an individual animal. While PCR is an excellent way to rule out KNOWN causes of disease, most diagnostic PCR assays are not designed to detect newly emergent disease. This is another reason to include histologic analysis of tissues in the diagnostic plan: by ruling out common diseases, a newly emergent cause for disease can be more quickly identified. The most difficult emergent diseases to recognize and identify are those that mimic known diseases. For retrospective analyses, DNA can also be extracted from tissue embedded in paraffin, however, this is offered by a more limited number of laboratories.

1 Toxicology:It is best to contact a toxicology laboratory, state laboratory, and/or a poison control center such as the ASPCA (http://www.aspca.org/apcc) for guidance. The appropriate sample for analysis is dependent on the type of toxin, among other variables. In the case of GI illness, source (food) and stomach contents should be saved. For heavy metal analysis, samples of liver and kidney should be collected, placed in separate plastic bags, and frozen until submitted. If a toxin is suspected but unknown, a necropsy should be performed and, in addition to histologic samples, liver, kidney, fat, stomach contents, and muscle samples should be frozen until submitted.

2 Serology:Serodiagnostic tests are tests performed on serum or plasma to detect either the presence of antibodies to a particular pathogen or the presence of circulating antigens from the pathogen itself. Both of these types of tests can be performed postmortem on serum obtained from a pooled blood source (e.g. heart, major veins). The significance of the result should be considered before this particular technique is used; few tests are validated for postmortem serum. Nonetheless, a positive titer is generally considered significant. See Chapter 4 for more details on serologic testing.