Hydrostatic Filtration Enables Large-Scale Production of Outer Membrane Vesicles That Effectively Protect Chickens against Gallibacterium anatis

Abstract

Gallibacterium anatis is a Gram-negative opportunistic avian pathogen representing an emerging threat to poultry meat and egg production worldwide. To date, no vaccine able to effectively prevent the morbidity associated with G. anatis infections has been developed yet. Our group previously reported that inoculation of different combinations of G. anatis outer membrane vesicles (OMVs), FlfA and GtxA-N proteins is effective in preventing lesions caused by G. anatis infections in layer chickens. Here we report the testing of the efficacy as vaccine prototypes of G. anatis OMVs isolated by hydrostatic filtration, a simple technique that allows the cost-effective isolation of high yields of OMVs. Layer chickens were immunized with OMVs alone or in combination with FlfA and/or GtxA-N proteins. Subsequent challenge with a heterologous G. anatis strain showed that immunization with OMVs alone could significantly reduce the lesions following a G. anatis infection. A second study was carried out to characterize the dose-response (0.25, 2.5 and 25 µg) relationship of G. anatis OMVs as immunogens, showing that 2.5 μg of OMVs represent the optimal dose to elicit protection in the immunized animals after a similar challenge. Additionally, administration of ≥2.5 μg of G. anatis OMVs induced specific IgY titers and possibly vertical transfer of immunity.

Sequence Analysis, Antibiogram Profile, Virulence and Antibiotic Resistance Genes of XDR and MDR Gallibacterium anatis Isolated from Layer Chickens in Egypt

“… 20 , 31 , 32 G. anatis is frequently retrieved from the genital and respiratory tracts of diseased poultry suffering from severe pathological lesions. 33 …”

Bacterial Outer Membrane Vesicles as a Versatile Tool in Vaccine Research and the Fight against Antimicrobial Resistance

“…Hydrostatic filtration dialysis (HFD) was originally developed by Musante et al as a way to quickly identify bacterial urinary tract infections by selectively enriching urine samples ( 95 ). The method was subsequently adapted and applied to the isolation of OMVs for vaccine development in several studies ( 96 – 98 ). HFD has been demonstrated to be a cost-efficient, simple, and reliable way of isolating OMVs, especially in cases where large numbers of OMVs are required, such as in vivo immunization trials.…”

“…HFD has been demonstrated to be a cost-efficient, simple, and reliable way of isolating OMVs, especially in cases where large numbers of OMVs are required, such as in vivo immunization trials. An additional advantage of HFD is that it offers a considerably consistent reproducibility from batch to batch, a characteristic rather crucial for vaccine development ( 98 ). As HFD has only recently been developed as an OMV isolation technique, the number of studies employing HFD for OMV isolation is currently too limited to offer a proper comparison between this and other OMV isolation methods previously mentioned.…”

Clarification of large-volume bacterial cultures using a centrifuge-free protocol

“…OMVs have gained significant attention due to their potential in preventing bacterial infections through vaccination (Mancini et al, 2020; Pors et al, 2016). A recent method employed for large‐scale isolation of OMVs from bacterial cultures relies on removal of bacterial cells using sterile filtration, OMV concentration using hydrostatic filtration (HF), and further concentration by Centrifugal Filter Units, which is often required to improve the yield of the OMVs (Antenucci et al, 2020). However, a less labour‐intensive clarification procedure is highly desirable for the production of OMVs.…”