Disease prevention against Gallibacterium anatis with special emphasis on outer membrane vesicles

PhD student: Toloe Allahghadry


The global emergence and spread of antimicrobial-resistant pathogens have become an increasing concern to public health. Besides affecting human healthcare, antimicrobial-resistant bacteria have also affected the livestock industry worldwide, which led to increased mortality and economic loss over the last decades.

Many farm animals, such as poultry, are regularly exposed to antibiotics and might act as a reservoir of resistant bacteria. Gallibacteriun anatis (G. anatis), a Gram-negative coccobacillus, is a normal microbiota in the upper respiratory and lower genital tracts of chickens. However, the bacterium acts as an opportunistic pathogen following penetration into the systemic circulation of chickens under disease-favouring conditions, which can lead to reduced egg production, increased mortality, and economic loss in poultry production systems.

Several studies on G. anatis strains have demonstrated a remarkably high prevalence of multidrug-resistant strains worldwide. Therefore, the widespread presence of multidrug-resistant G. anatis isolates in different poultry production systems calls for efficient non-antibiotic prophylactic measures. One of the most effective strategies to prevent and control antimicrobial resistance (AMR) is to avoid the need for treatment by vaccination. Outer membrane vesicle (OMV) vaccines have broadened the horizon of effective prophylactic measures against various infectious pathogens.

There is a great potential in self-adjuvanting OMVs as a genetically programmable vaccine platform to induce broad immunity across strains.

Purpose of the project

The purposes of the PhD project were to investigate:

  1. Occurrence, genetic diversity, AMR and presence of potential vaccine candidates against anatis strains in chicken production systems in Iran.
  2. Clarification of large-scale bacterial cultures using a centrifuge-free single-step protocol to improve production of OMVs.
  3. Exogenous loading of surface-exposed antigens on OMVs using Plug-and-Display technology to develop a novel and flexible vaccine platform technology based on immunogen-enriched OMVs for optimal exposure the immune system.


Several genotypes of G. anatis were identified in chicken production systems. Identical clones were also shared among chickens within and between different farms, indicating the clonal presence of G. anatis in epidemiologically unrelated farms. The presence of multidrug- resistant G. anatis isolates calls for non-antibiotic prophylactics. Further investigations into alternative means to prevent and control G. anatis strains led to the characterization of three immunogens as potential vaccine targets, including the GtxA, Gab_1309, and Gab_2312 proteins. Following the gene sequence analysis of the selected immunogens, GtxA protein toxin was suggested for future vaccine development due to a conserved sequence identified across a range of epidemiologically independent strains.

In a separate study, a reliable and reproducible centrifuge-free filtration system was developed for more efficient clarification of bacterial cultures, maximizing the yield of the OMVs isolated (Universal applicability). The consistent flow rate without filter clogging, sterility assurance, and increased product yield compared to the conventional method indicate the high efficiency of the system developed, regardless of the volume of culture harvested.

Finally, a novel and flexible vaccine platform technology was developed based on bioengineered OMVs as a promising vaccine candidate against infectious diseases. Successful development of Catcher-OMVs system provided a universal SpyCatcher-OMV platform to which a Spy-tagged antigen(s) could be bound (Catcher-OMV system). In the light of the Catcher-OMV system, there is no limitation for exogenous loading of surface-exposed antigens on OMVs.

Future perspectives

It was indicated that the toxin gene gtxA appears to be a potential vaccine candidate against G. anatis strains. It is essential to confirm that the vaccine candidate introduced is indeed able to reduce the severity of G. anatis infection in chickens. Accordingly, a future investigation is suggested to be performed in vivo to evaluate the immunogenic efficiency of the selected vaccine candidate through vaccination of chickens by the GtxA protein toxin proposed.

The results achieved in production of OMVs highlight the importance of the centrifuge-free filtration system developed. In the future, further testing of the filtration system with different bacteria is suggested to be performed.

A follow-up study applying the Catcher-OMV system should be performed, in which the sfGFP should be replaced by a target antigen(s), to be loaded on the exterior of OMVs for optimal exposure to the immune system. Additionally, to evaluate the performance and immunogenic efficiency of the tailor-made vaccine developed, in vivo validation will be needed.