Histopathology can provide a strong framework to establish the real causality of a clinical problem.
J. Segalés
Histopathology: an added value tool to provide consistency to the diagnostic investigation
Apart from those laboratory tests specifically focused on the determination of pathogens or toxins, histopathology can provide a strong framework to establish the real causality of a clinical problem. Said in other words, detection of a particular pathogen or toxin must be consistent with the clinical, epidemiological and gross lesion observations, and histopathological analyses can definitively support such consistency.
A good example of microscopic assessment usefulness would be the detection of a virus by PCR in a respiratory problem in which the typical histological lesions caused by this agent are not present in the lung (assuming the pig or group of pigs are representative of the observed disease). Such scenario should prompt to recapitulate on the suspected diagnosis and investigate further.
The swine veterinarian should consider histopathology as a very valuable tool, at least as important as PCRs, bacterial isolation or antibody detection techniques. Microscopic analyses allow morphological diagnoses that reinforce or contradict what has been established by means of clinical, epidemiological or other laboratory results, therefore contributing to establish the likely etiopathogenesis of the clinical problem. This overall information is important, since is a kind of predictor on how the control and prevention measures implemented to counteract the disease may work or not.
Sample collection for histopathology (and for other laboratory tests) is relatively easy but demands certain disease pathogenesis knowledge as well as some technical skills:
- Specimens must be collected from fresh carcasses (ideally recently dead or euthanized pigs).
- Tissues be immediately fixed by immersion in 10% buffered formalin.
- Submission of refrigerated samples for histopathological analyses is not recommended, since certain degree of autolysis occur.
- Also, frozen samples imply multiple artifacts that jeopardizes microscopic interpretation.
- Tissue sample size must be relatively small (or at least thin to favour fixation).
- Samples should include grossly affected portions as well as pieces of presumably normal tissue (Figure 1).
- In case of intestine samples, it is important to longitudinally open the intestine to ensure that formalin is in direct contact with the enteric mucosa.
- In contrast, a big organ such the brain is preferred being fixated entire or one half; the reason for this procedure is to ensure that brain topography is maintained during fixation, and then the pathologist can select the right portion to try to establish the most likely diagnosis.
Figure 1. A. Recommended general sampling in a lung, taking pieces that include normal as well as damaged areas. B. In case of focal lesions, normal and damaged area should also be taken, not only damaged one.
Importantly, besides the plain histopathological analysis (based on the haematoxylin and eosin stain, Figure 2), other pathological additional tests may help in seeking for etiological agents.
Figure 2. Haematoxylin and eosin stain in a normal (A) and damaged (B) lung. The lung in B is affected by a fibrino-necrotizing pleuropneumoniae caused by Actinobacillus pleuropneumoniae.
The most used ones are immunohistochemistry (Figure 3) and in situ hybridization (Figure 4), which allow detecting pathogens at the site of action, supporting their role in the clinical and pathological context. Besides, there are other techniques (histochemical stains), which are not sufficient to establish a specific aetiology, but able to give indications about the cause (e.g., Groccot stain to detect fungi or Gram stain to detect gram-positive or -negative bacteria).
Figure 3. Immunohistochemistry to detect porcine reproductive and respiratory syndrome virus (PRRSV) (A) and swine influenza virus (B). Note that presence of viral antigen (brown staining) is found in damaged areas.
Figure 4. In situ hybridization to detect porcine circovirus 2 (PCV-2) in a lung affected by interstitial pneumonia.
In summary, the swine veterinarian, besides his/her own clinical, epidemiological and gross lesion investigations, has a variety of analytical possibilities, in which histopathology can play a vertebral role. Effective communication between clinicians and pathologists enhances diagnostic capabilities, leading to earlier and more efficient resolution of clinical problems.
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