One of the major bacterial infectious diseases in the poultry industry is avian pathogenic Escherichia coli (APEC), which causes colibacillosis in chickens. To develop a novel nucleic acid-free bacterial ghost (BG) vaccine against the O78:K80 serotype of APEC, in this study we constructed a plasmid that harbored E-lysis and S nuclease (SNUC). Following the expression, the O78:K80 bacteria lost all of their cytoplasmic content and nucleic acids by enzymatic digestion. The functionality of these two proteins in the production procedure of bacterial ghosts was confirmed by monitoring the number of colonies, scanning electron microscopy imaging, gel electrophoresis of genomic DNA, and qPCR on the plasmid content of bacterial ghosts. The protective efficacy of the ghost vaccine generated from O78:K80 serotype of APEC was tested in chickens by injection and inhalation routes and compared with that in chickens that received the injection of a killed vaccine. The O78:K80 BG vaccine candidate, used as injection and inhalation, in comparison with the killed vaccine, triggered higher proinflammatory cytokine expression including IL-6, IL-1β, and TNFSF15; a higher level of antibody-dependent humoral (IgY and IgA) and cellular immune responses (IFNγ and lymphocyte proliferation); and lower lesion scores. According to the results of this study, we suggest that the bacterial ghost technology has the potential to be applied for the development of novel vaccines against avian colibacillosis. This technology provides an effective and reliable approach to make multivalent vaccines for more prevalent APEC strains involved in the establishment of this infectious disease in the poultry industry.

Mycoplasmosis is one of the most important diseases in the poultry industry. Its causative agent, mycoplasma has various species, which two of them, Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most important species. Due to the enormous losses in the production farms of industrial poultry, achieving a rapid, accurate and definite diagnosis of mycoplasma is of great importance. An early and definite diagnosis can guarantee the farm management on keeping herd health. In many countries such as Iran, the disease and its complications have still remained as a serious problem. Given this issue, we decided to identify the mycoplasma infection from broiler poultry flocks through culture method. 150 carcasses of broiler chicken belonging to 50 broiler flocks were sampled in which the signs of air sacs involvement and secretions in the airways, trachea and bronchi were seen. Samples taken from trachea, palatine cleft, nasal passages and air sacs, were cultivated into PPLO liquid medium using membrane filters (0.45 micron). They were incubated at 37 °C and were examined for pH (color) changes for every 48 hours. During the first 24 hours after cultivation, every color change to yellow or dark visible with the bare eye was considered as bacterial contamination, therefore, the contaminated samples were removed from the incubator. The color change in the liquid media was compared with the uninoculated medium as negative control. If a color change was observed in the liquid media after 48h, subculture was done in the PPLO agar. The plates were incubated at 37 °C for 14 days. They were examined for mycoplasma colonies using a microscope with magnification of 10 in every other day. The results showed that out of 150 samples obtained from 50 broiler flocks, 16 (10.66%) were positive for mycoplasma, while in terms of contamination, 4 flocks (8%) were positive. The contamination of positive cultures was finally confirmed through PCR method with universal primer.