bis(3--5-)-cyclic-diguanylic-acid and Poultry-Diseases

In an effort to explore strategies to control Clostridium perfringens, we investigated the synergistic effect of a ubiquitous bacterial second messenger 3',5'-cyclic diguanylic acid (c-di-GMP) with penicillin G in a broiler challenge model. All chicks were inoculated in the crop by gavage on d 14, 15, and 16 with a mixture of 4 C. perfringens strains. Birds were treated with saline (control group) or 20 nmol of c-di-GMP by gavage or intramuscularly (IM) on d 24, all in conjunction with penicillin G in water for 5 d. Weekly samplings of ceca and ileum were performed on d 21 to 35 for C. perfringens and Lactobacillus enumeration. On d 35 of age, the IM treatment significantly (P < 0.05) reduced C. perfringens in the ceca, suggesting possible synergistic activity between penicillin G and c-di-GMP against C. perfringens in broiler ceca. Moreover, analysis of ceca DNA for the presence of a series of C. perfringens virulence genes showed a prevalence of 30% for the Clostridium perfringens alpha-toxin gene (cpa) from d 21 to 35 in the IM-treated group, whereas the occurrence of the cpa gene increased from 10 to 60% in the other 2 groups (control and gavage) from d 21 to 35. Detection of β-lactamase genes (blaCMY-2, blaSHV, and blaTEM) indicative of gram-negative bacteria in the same samples from d 21 to 35 did not show significant treatment effects. Amplified fragment-length polymorphism showed a predominant 92% similarity between the ceca of 21-d-old control birds and the 35-d-old IM-treated c-di-GMP group. This suggests that c-di-GMP IM treatment might be effective at restoring the normal microflora of the host on d 35 after being challenged by C. perfringens. Our results suggest that c-di-GMP can reduce the colonization of C. perfringens in the gut without increasing the selection pressure for some β-lactamase genes or altering the commensal bacterial population.

Topics: Adjuvants, Immunologic; Animal Feed; Animals; Anti-Bacterial Agents; Bacterial Toxins; Cecum; Chickens; Clostridium Infections; Clostridium perfringens; Colony Count, Microbial; Cyclic GMP; Enteral Nutrition; Enteritis; Injections, Intramuscular; Male; Penicillin G; Polymerase Chain Reaction; Poultry Diseases

Necrotic enteritis (NE) caused by netB-positive strains of Clostridium perfringens is an important disease of intensively-reared broiler chickens. It is widely controlled by antibiotic use, but this practice that has come under increasing scrutiny and alternative approaches are required. As part of the search for alternative approaches over the last decade, advances have been made in understanding its pathogenesis but much remains to be understood and applied to the control of NE. The objective of this work was to assess the effect on virulence of mutation of the cyclic-di-GMP signaling genes present on the large pathogenicity locus (NELoc-1) in the tcp-encoding conjugative virulence plasmid, pNetB. For this purpose, the diguanylate cyclase (dgc) and phosphodiesterase (pde) genes were individually insertionally inactivated and the two mutants were subsequently complemented with their respective genes. Southern blotting showed that a single gene insertion was present. Mutation of either gene resulted in almost total attenuation of the mutants to cause NE in experimentally-infected broiler chickens, which was fully restored in each case by complementation of the respective mutated gene. Production of NetB-associated cytotoxicity for Leghorn male hepatoma (LMH) cells was unaffected in mutants. We conclude that the cyclic-di-GMP signaling system is important in controlling virulence in a NE C. perfringens strain and might be a target for control of the disease.

Topics: Animals; Chickens; Clostridium Infections; Clostridium perfringens; Cyclic GMP; Enteritis; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Intestines; Mutation; Poultry Diseases