SAMPLING

In HISTOPATHOLOGY , the time elapsed since death and autolysis are the main impediments for a correct evaluation of the lesions and the detection of infectious agents in the enteric cells. For example, 20-30 minutes after death there is a diffuse detachment of enterocytes from the villus tips due to autolysis, which makes the histopathological diagnosis of ETEC and I. suis impossible. At two hours postmortem, all the tissue from the middle to the tip of the villi becomes eosinophilic without distinction between cells due to autolysis. Since most samples arrive at the laboratory the day after they are collected, the exclusive shipment of intestinal samples would significantly limit, or even prevent, a proper histological evaluation. As a consequence, it is essential that, if you are not going to send live animals to the laboratory, you collect the intestinal samples right after the euthanasia of the piglets. The fragments of intestine must have 2 to 3 cm in length and proceed from 2 portions from the ileum, between 4 and 5 from the jejunum, one from the cecum, one from the proximal colon and 2 from the spiral colon and be fixed in formalin (10%) in plastic bags or containers. Mesenteric lymph nodes and liver fragments should also be sent. All these samples will be used for histopathological examination and, in some cases, also for immunohistochemistry (PED, TGE, Rotavirus) or in situ hybridization (ISH).

In addition to formalin-fixed samples, it is important to submit fresh samples for bacteriology, virology, and molecular and biological analyses . Fragments should be 10 to 15 cm in length from the ileum and jejunum and the remaining portions of the cecum and spiral colon and may be shipped in a plastic bag. Mesenteric lymph nodes and liver fragments must be sent in separate bags to avoid contamination.

The histological evaluation of the intestinal sections mentioned above should make it possible to limit the possible agents involved in the process and, in some cases, define the presence of infectious agents. For example, villous atrophy can be caused by coccidia (I. suis), Rotavirus, GETv, PEDv, Deltacoronavirus or even C. perfringens type A. If the samples belong to piglets older than five or six days, we can find sexual or asexual forms of I. suis in the cytoplasm of villus-tip enterocytes. If coccidia are not found, immunohistochemistry can be performed to detect the presence of Rotavirus or Coronavirus viral antigens in the enterocytes. In almost all cases of ETEC, histologic examination will allow detection of a myriad of coccobacillary organisms attached to the surface of the enterocyte at the base or lateral wall of the villi. C. perfringens type C will cause a transmural fibrinonecrotic and hemorrhagic lesion in a segmental distribution in the small intestine. C. difficile is the only infectious agent that will cause injury to the large intestine. Mesocolon edema is often observed. The main findings in cases of C. difficile infection are the reduction of goblet cells and a large infiltration of neutrophils in the mucosa (of the colon that, in some cases, rupture the epithelium like a volcanic eruption. C. perfringens type A is the most difficult to determine by histopathology, since it can cause villous atrophy, but the fact that this lesion is not found does not rule out this possibility. (Roberto MC Guedes)