History

An approximately 2-month-old 31.8-kg (70-lb) castrated male feeder pig that died was submitted for necropsy examination at the Diagnostic Center for Population and Animal Health at Michigan State University. The pig was raised on a farm for which porcine circovirus type 2 (PCV-2) infection had been reported in the 2 years before the pig's death; pigs on the farm had received appropriate vaccines since that time. Recently, a group of feeder pigs became ill; the producer suggested the illness was possibly due to heat stress.

Gross Findings

The pig was in fair nutritional condition (body condition score, 2 [scale, 1 to 5]) with mild dehydration and mild autolysis. The mucosa of the ileum was diffusely thickened and corrugated. The large intestine was distended with bloody fluid and sand, and the large intestinal mucosa was thickened and diffusely covered with a yellowish-gray pseudomembrane that tightly adhered to the mucosa (Figure 1).

Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page →

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Photograph of the opened spiral colon of a feeder pig from a herd with a recently increased morbidity rate. Notice the marked thickening of the mucosa and the multifocal brown-gray necrotic pseudomembrane along the surface (highlighted in the inset).

Citation: Journal of the American Veterinary Medical Association 248, 8; 10.2460/javma.248.8.897

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Histopathologic Findings

Tissue samples of the small intestine and colon were examined histologically. In the sections of small intestine, in particular those of the ileum, there was marked crypt proliferation characterized by variably dilated, tortuous crypts lined by tall columnar, basophilic, mitotically active epithelium (21 mitoses/10 hpfs) that was frequently devoid of goblet cells. Intraluminal crypt cell debris was also noted, and occasionally there was segmental necrosis of the crypt lining epithelium. Additionally, there was multifocal necrosis of the tips of the villi, with necrotic debris attached to the luminal surface.

Sections of colon had similar crypt epithelial proliferation (Figure 2). Additionally, corresponding to the pseudomembrane observed grossly, the superficial third of the mucosa was diffusely necrotic and had been replaced by eosinophilic cellular debris. Within the junction area between necrotic and viable mucosa, there were numerous monomorphic (1 × 4 μm) bacillary bacteria. Multiple vessels with fibrin thrombi were detected in the subjacent lamina propria. The deeper portion of the lamina propria had mild lymphoplasmacytic infiltration. Mild neutrophilic infiltration and vasculitis were present in the submucosa.

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Photomicrographs of sections of the spiral colon from the pig in Figure 1. A—Notice the marked thickening of the mucosa with hyperplastic crypts and necrotic surface (asterisks). Multiple fibrin thrombi are evident in the junction area between viable and necrotic tissue (arrows). H&E stain; bar = 1.0 mm. B—Numerous gram-negative bacilli are present at the interface between the necrotic and viable portion of the mucosa (arrow). Gram stain; bar = 200 gm. C—Numerous argyrophilic bacilli are visible in the apical cytoplasm of the crypt epithelium (arrow). Warthin-Starry stain; bar = 100 gm. D—Immunohistochemical staining reveals that the bacilli in the apical cytoplasm of the crypt epithelium are Lawsonia intracellularis. Rabbit polyclonal antibody against L intracellularis and amino-ethyl-carbazol chromogen (red); bar = 200 gm.

Citation: Journal of the American Veterinary Medical Association 248, 8; 10.2460/javma.248.8.897

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Additional Test Results

In gram-stained sections of colon tissue, numerous gram-negative bacilli (approx 1 × 4 μm) were mainly located deep in the necrotic debris, at the border with the viable tissue (Figure 2). Additionally, multiple gram-positive bacilli were observed in the necrotic debris along the luminal surface of the villi. In Warthin-Starry–stained sections of colon tissue, there were numerous argyrophilic bacilli in the apical cytoplasm of the crypt epithelium. These bacilli were immunoreactive with a rabbit polyclonal antibody specifically against Lawsonia intracellularis. Bacterial culture of samples of the colon yielded Salmonella enterica, but not Brachyspira spp. Additionally, PCR testing for PCV-2 was performed on pooled specimens of spleen, lymph node, and lungs; the results were positive.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: severe, diffuse, proliferative, and necrotizing enterocolitis.

Case summary: proliferative and necrotizing enterocolitis in a pig resulting from coinfection with L intracellularis and S enterica.

Comments

Given the results of gross examination of the carcass and findings of histologic examination, histochemical and immunohistochemical analyses, and bacterial culture of selected tissue specimens, a diagnosis of proliferative and necrotizing enterocolitis attributable to coinfection with L intracellularis and S enterica was made for the pig of the present report. Lawsonia intracellularis is an intracellular gram-negative, argyrophilic bacterium that causes porcine proliferative enteropathy as well as proliferative enteropathy in other species, including rats, hamsters, guinea pigs, dogs, whitetailed deer, horses, primates, ostriches, and emus.^1–3 This disease has a worldwide distribution and continues to be a cause of major economic loss in swine herds.^3,4

In pigs, L intracellularis infection develops in animals older than 4 weeks of age; thus, this condition is a postweaning disease that is typically detected during the grower phase.^1,3 Lawsonia intracellularis preferentially infects, and localizes in, the apical cytoplasm of the crypt epithelium of the intestinal tract,^2,3 as revealed in tissue samples from the pig of this report following Warthin-Starry staining and immunohistochemical analysis for L intracellularis. Lawsonia intracellularis induces the upregulation of host genes involved in cell cycle regulation, cell differentiation, and cell proliferation, which is the likely mechanism responsible for the hyperproliferation of crypt epithelial cells detected 3 to 5 days after infection.^2,5 In general, gross lesions are confined to the intestinal tract (mainly to the mucosa of the ileum and occasionally that of the large intestine) and generally characterized by thickening of the intestinal wall.^2,3 Early lesions consist of focal, small, raised areas in an otherwise normal mucosa, whereas advanced lesions are characterized by confluent mucosal nodules and folds.³ Histologically, the affected areas of small and large intestines have loss of the normal mucosal architecture with replacement by tortuous crypt glands. Cells lining hyperplasic glands are generally described as crowded and have elongated and either vesicular or dark-staining basal nuclei.³ In affected tissue samples, the cell mitotic index is reported to be high,³ as it was in the case described in the present report. Shedding of infected epithelial cells into the intestinal lumen acts as the source of infection for other pigs via the fecal-oral route.³ Subclinical and chronic infection is common, and outbreaks of enteritis caused by L intracellularis infection are considered to be stress related.⁴ Environmental and other stressors, including transportation, sorting, or commingling, often precipitate outbreaks.^4,6

In the case described in the present report, severe superficial mucosal necrosis was also present in the colon, and S enterica was cultured from the colon. Salmonellae are gram-negative enteroinvasive bacteria.¹ All known serotypes of S enterica are pathogenic and causes of important zoonotic and nosocomial infections.^1,7 Salmonellosis can be associated with acute and chronic diarrhea and death in numerous animal species, including humans.¹ In pigs, salmonellosis can result in peracute septicemia and acute and chronic enterocolitis.¹ Although peracute Salmonella septicemia is usually fatal in animals of 1 to 6 months of age,¹ affected pigs may completely recover and some can remain as carriers and intermittent shedders for several months.⁸ Following oral ingestion, Salmonella organisms adhere to the apical surface of the intestinal epithelium to begin the process of colonizing the intestines.^1,9 In the acute enteric form of the disease, mucosal adherence and colonization lead to mucosal necrosis mediated by bacterial toxins.¹ Adhered bacteria invade the enterocytes and M cells, resulting in the extrusion of infected epithelial cells into the intestinal lumen with subsequent villus blunting and loss of absorptive surfaces.⁹ Salmonellae are motile bacteria, and invasion of the lamina propria with direct effects on the vasculature, which result in thrombosis, occurs in the chronic enteric form of the disease, leading to foci of necrosis known as button ulcers.¹ The colon tissue samples from the pig of this report had severe superficial mucosal necrosis with numerous intralesional gram-negative bacteria, and multiple vessels in the lamina propria had fibrin thrombi. On the basis of these histopathologic findings and the result of bacterial culture, S enterica was considered to be a major contributor to the mucosal necrosis. Gram-positive bacilli present along the luminal surface were interpreted as early postmortem clostridial overgrowth and thus regarded as clinically unimportant.

Although the pathological features of coinfection with S enterica and L intracellularis have not been described in pigs, previous studies have identified swine cases of coinfection with these organisms via PCR testing, bacterial culture of fecal samples, and serological tests.^4,7,10 In particular, the simultaneous detection of L intracellularis and S enterica is not uncommon; up to a third of swine herds with a history of diarrhea that tested positive for salmonellae have been determined to be coinfected with L intracellularis.⁴ Enteric health problems predispose pigs to subclinical salmonellosis because of a disturbance of the ecological balance of the gastrointestinal flora.¹¹ The risk for shedding of salmonellae and onset of enteric disease is increased in pigs that have detectable serum antibodies against L intracellularis and porcine reproductive and respiratory syndrome virus during the fattening period.^8,11 Thus, on the basis of the time course of lesion development, proliferative enteritis associated with L intracellularis infection was likely the primary process in the pig of the present report, and necrotizing enteritis attributable to Salmonella infection was secondary.

Diarrhea resulting from PCV-2–induced enteritis is sometimes the only expression of PCV-2–associated disease in pigs.¹² The pig of the present report was raised on a farm that had previous reports of PCV-2 infection, and a pool of tissue samples collected at necropsy tested positive for this virus. Nevertheless, there was no evidence of the characteristic microscopic lesions in lymphoid and enteric tissues typically associated with PCV-2 infection¹² to suspect a confounding PCV-2 infection in this animal.