Search for Lawsonia intracellularis and Bilophila wadsworthia in malabsorption-diseased chickens

Abstract

Proliferative enteropathy is an important enteric disease caused by Lawsonia intracellularis. A wide range of host species can be infected by the same bacterium, yet the clinico-pathologic features among these hosts remains almost identical. The disease has been recognized regularly among ratites, but not in other avian families, such as galliforms, even though these suffer uncharacterized enteric conditions. Fresh ileum-colon contents were obtained from 228, 3- to 8-week-old chickens with enteric disease, kept at 14 large commercial farms in the southern USA. DNA was extracted from each sample and subjected to polymerase chain reactions (PCR) with primers specific to eubacterial DNA, L. intracellularis, and Bilophila wadsworthia. All chicken samples were positive for eubacterial DNA, 29 chickens (13%) were positive for B. wadsworthia DNA, and none were positive for L. intracellularis DNA. Given the ubiquitous nature of L. intracellularis, we consider it likely that some avian families do not carry the necessary mechanism for L. intracellularis viability. Bilophila wadsworthia appears to be a consistent member of the colonic flora of some host animals. Neither bacterium appears to be associated with malabsorption syndromes in chickens.

Proliferative enteropathy is an important enteric disease with prominent intestinal mucosal hyperplasia, caused by the obligate intracellular Gram-negative bacterium Lawsonia intracellularis (1). A wide host species range, including omnivores, herbivores, and carnivores can be infected by the same bacterium, yet the clinico-pathologic features among these hosts remains almost identical. The disease and its agent are widely diagnosed among pigs, horses, and hamsters in North America and elsewhere (1,2). The agent is considered as an obligate pathogen, that is, it has not been recognized in hosts in the absence of at least minor lesions closely associated with the site of the agent (1,3). To a lesser extent, the disease has been recognized among captive ratites (emu and ostriches), but not in other avian families, such as galliforms (2,4). In certain other host species, namely rats and rhesus macaques, only a single outbreak of the disease has been confirmed in the past 30 y, each time in a laboratory animal colony setting in Europe or USA, respectively (5,6); even though enteric disease among other groups of these animals are regularly investigated. Prominent clinical signs of proliferative enteropathy are diarrhea and poor weight gain, and similar clinical syndromes (malabsorption) are common in commercial chicken flocks in North America and elsewhere. The exact etiology of these remains uncharacterized, but certain strains of avian reovirus may play an important role (7). Bacterial investigations of malabsorption syndromes in chickens have not elucidated any specific agents, but neither of the subjects of this study has been targeted previously (8).

The closest bacterial relative to L. intracellularis is Bilophila wadsworthia, an anaerobic, free-living bacterium with 92% genomic DNA homology. It is a common, non-pathogenic resident in the colon of humans and pigs (9,10), but has been associated occasionally with appendicitis lesions (9). The aim of this study was to investigate the possibility that L. intracellularis or B. wadsworthia may play a role in malabsorption syndromes in galliforms.

A total of 228 chickens (Gallus domesticus), 3 to 8 wk of age, were collected from 14 large commercial farms in the southern USA. Group history, clinical signs of diarrhea, and poor growth rates were established, then efforts were made to select currently affected chickens from among the group examined. Selected chickens (at least 5 per group) were euthanized and subjected to a diagnostic autopsy. The content from each fresh ileum and colon was collected by forcible pressure. Suspensions of each ileum-colon contents were prepared by vortexing 2 g with 5 mL of a commercial lysis buffer, then incubated at 70°C to release bacterial DNA. The lysis buffer (high-strength, chaotropic guanidium salts and detergents) and subsequent steps formed part of a DNA extraction kit technique designed for fecal-intestinal materials (19082 and 51504, respectively; QIAGEN, Valencia, California, USA). This kit also incorporates a specific polysaccharide mixture at an absorption step, designed to remove the numerous polymerase chain reaction (PCR) inhibitors from the fecal-intestinal material, and is considered superior to other commercial or non-proprietary methods for DNA extraction from this source (11).

Each DNA extraction sample was subject to PCR for eubacterial DNA, L. intracellularis DNA, and B. wadsworthia DNA using specific PCR primers and reaction cycle parameters, as previously described (10,12). The purpose of the initial eubacterial DNA test (primers p11E and p13B, specific for all known eubacterial genera, (13)) was to establish that the bacterial DNA extraction from each sample was successful and to eliminate one possible source of false negatives. The standard PCR test used for L. intracellularis incorporates a within-sequence nested primer set approach for maximum, reliable sensitivity (2,12). The DNA from both L. intracellularis and B. wadsworthia derived from defined laboratory strains of each organism (1,10), were available and incorporated into each PCR batch as positive controls, consistently leading to the expected PCR products of 270 and 207 base pairs (bp), respectively.

Autopsies of the chickens confirmed the presence of diarrhea and poor nutritional condition consistent with malabsorption. All chicken ileum-colon content samples were positive for eubacterial DNA, 29 (13%) were positive for B. wadsworthia DNA and none were positive for L. intracellularis DNA. Both L. intracellularis and B. wadsworthia are highly fastidious organisms (1,9), therefore, isolation cultures were not attempted. Specific lesions due to proliferative enteropathy were not noted during the autopsy and subsequent histology of any of the chickens. Silver stains and other histologic techniques aimed at detecting L. intracellularis were not performed, as these lack sensitivity compared to PCR (14).

The exact cause of malabsorption syndromes in chickens is not yet clear, although reoviruses are thought to play an important role (7,8). Despite a targeted search over several farms across a wide area, L. intracellularis was not identified as contributing to the malabsorption syndrome of chickens from any of these affected farms. As this was considered the most likely site of infection for this potential host species, we suggest that it is likely that chickens are not routinely infected by this pathogenic bacterium. Surveys of other host species, such as farmed pigs, conducted using similar fecal-PCR methodology regularly detect L. intracellularis-positive cases (15). Similarly, in a previous study, 1-day-old chickens did not develop any detectable infection following exposure orally to L. intracellularis (16). Given the ubiquitous nature of L. intracellularis, and its occasional occurrence in captive ratites, we consider it likely that some avian families do not carry the necessary mechanism for L. intracellularis viability, rather than lack access to or possess any innate immunity to the agent. The wide range of hosts of L. intracellularis and its universal causation of proliferation of immature epithelial cells in the intestinal crypts of all these species, clearly argues that some form of universal epithelial cell receptor and cell cycle pathway is affected during the course of intracellular infection, but that this pathway is not active among chickens. Similarly, L. intracellularis infection has never been identified in humans, despite ample exposure, particularly among pig farmers and veterinarians; therefore, it is not considered a zoonosis. Unfortunately, the actual cell receptor and downstream effects are not yet known for this agent. The pathogenesis of the disease in its early stages (1,3) suggests that L. intracellularis may evoke some interference with p27 cyclin kinases, responsible for the differentiation of immature crypt cells into mature ones.

Bisophila wadsworthia appears to be a consistent member of the anaerobic colonic flora of some animals, including humans, pigs, and chickens. There is a marked lack of controlled data regarding the challenge exposure effect of this agent on animals (9). Therefore, it is not clear as to the exact role of B. wadsworthia, if any, in the enteric conditions among chickens or other animals, despite its prevalence among these malabsorption-affected chickens. Direct bacterial investigations of malabsorption syndromes in chickens have not elucidated any specific agents (8). Certain strains of avian reovirus appear to be capable of initiating this syndrome, but the exact strains and their virulence factors or secondary bacterial agents are not yet clear (7).