Association of Beta2-PositiveClostridium perfringensType A With Focal Duodenal Necrosis in Egg-Laying Chickens in the United States

Abstract

Focal duodenal necrosis (FDN) is a poorly understood intestinal disease of egg layers, and has been associated with drops in egg production and decreased egg weights. The etiology of this disease is still unknown, but the condition has been associated with Clostridium colinum and Clostridium perfringens. In order to investigate the etiology, duodenal samples were taken from hens with FDN. The hens originated from table egg layer farms in three states. The samples were examined by histopathology, bacteriology, and immunohistochemistry. Macroscopically, all samples contained focal to multifocal, variably sized, reddened or brownish gray areas of mucosal erosion. Histopathology revealed mild to severe heterophilic and lymphoplasmacytic enteritis with loss of enterocytes at the villous tips, luminal fibrinonecrotic exudate, and variable numbers of Gram-positive and Gram-negative rod-shaped bacteria within the lesions in 16/30 samples. Clostridium perfringens was isolated by anaerobic bacteriology from 4/13 samples that had characteristic microscopic lesions of FDN. Polymerase chain reaction (PCR) revealed that all four isolates were Type A C. perfringens, positive for beta2 gene and negative for necrotic enteritis toxin B and enterotoxin genes. PCR for Clostridium colinum applied to DNA extracted from frozen intestinal samples yielded negative results in 14/14 duodenal samples. Immunohistochemistry (IHC) for 7C. perfringens, alpha and beta2 toxins stained a few to numerous long rod-shaped bacteria present in the lesions. IHC for alpha and beta2 toxins also stained enterocytes at the villous tips, inflammatory cells in the lamina propria, as well as degenerated and sloughed enterocytes present within the luminal exudate. These findings suggest that C. perfringens may play a role in the development of FDN. Experimental challenge studies with these isolates still need to be performed in order to reproduce the disease and fulfill Koch's postulates.

Toxinotyping of Clostridium perfringens in Poultry from Necrotic Enteritis Cases

“…Nyrah et al (2017) detected total 66 isolates of C. perfringens and showed 100 percent positivity towards cpa toxin while 13 isolates (19.69%) showed positivity for cpb2 toxins. Recently, it was speculated that cpb2 positive C. perfringens may cause focal duodenal necrosis in egglaying chickens (França et al, 2016). The results of cpb2 in their study was in agreement with the present findings with similar agreement.…”

Disease surveillance in England and Wales, June 2019

“…The aetiology of FDN remains uncertain but the disease is associated with the presence of Clostridium colinum infection or with toxin-producing Clostridium perfringens strains. 2 The disease has been reported in layer chickens under a variety of management types, and can result in decreased egg production and lower egg weights, and may represent a mild form of necrotic enteritis which is more oten seen in younger birds.…”

“…FDN has been regarded as one of the top ive diseases afecting layer chickens in the USA, 2 but has rarely been reported in Great Britain.…”

Complete Genome Sequence of Clostridium perfringens LLY_N11, a Necrotic Enteritis-Inducing Strain Isolated from a Healthy Chicken Intestine

“…Computational analysis revealed that strain LLY_N11 contained 20 toxin genes, including alpha-toxin, alpha-clostripain, enterotoxins (EntA, EntB, and EntD), hemolysin, kappa-toxin, mu-toxin, and beta2-toxin. Alpha-toxin (encoded by cpa ) was the most toxic extracellular enzyme that hydrolyzed important constituents of eukaryotic cell membranes ( 11 , 12 ), while beta2-toxin was found to be associated with intestinal disorders in chickens and other food animals ( 13 , 14 ). Based on the toxin gene types ( 1 ), C. perfringens strain LLY_N11 is a new strain of C. perfringens type A due to absence of beta, iota, and epsilon toxin.…”