Detection of necrotic enteritis risk through non-invasive monitoring of Clostridium perfringens in feces

Necrotic enteritis (NE), caused by the gram-positive, anaerobic bacterium, Clostridium perfringens, results in an estimated $6 billion in annual economic losses to the global poultry industry. C. perfringens is part of the normal microflora of the poultry gastrointestinal tract, but damage to the intestinal epithelium can lead to increased cell proliferation and production of toxins which gives rise to disease. Conventional methods to diagnose NE are invasive and are typically performed after the disease has manifested. In a pen trial using a necrotic enteritis model, we demonstrate that non-invasive monitoring in feces can detect an increase in average C. perfringens counts that correlates with higher lesion scores and reduced body weight gain in birds with NE. This was achieved using a C. perfringens-specific fluorescence in situ hybridization (FISH) probe and a high throughput platform (PIPERTM) for concentrating, imaging, and automated counting of labeled cells. The assay detects all tested strains of C. perfringens while excluding closely related bacteria, including other Clostridium species commonly found in poultry feces. The counts by the PIPER assay show a linear log-log relationship with the counts obtained by conventional plating of spiked fecal samples. Furthermore, fecal samples can be stored for up to 72 h without a dramatic loss in C. perfringens detection on this platform using the recommended sample collection and storage conditions. This non-invasive assay could open new opportunities for early identification of NE risk as well as for quantifying intervention efficacy.