Evaluation of the Efficacy of Humoral Immunity Response of Killed Oil Adjuvant Escherichia coli Vaccine in Layer Chicken against Avian E. coli Serotype O78 Infection

1. Introduction

Colibacillosis is a disease caused by infection with a strain of Escherichia coli ( 1 ). Escherichia coli is a gram-negative, rod-shaped bacterium that is normally found in the intestine of poultry and other vertebrates. Though many E. coli are not pathogenic, some have acquired virulence factors, greatly increasing their capacity to cause disease ( 2 ). Colibacillosis results in a localised or systemic infection caused by avian pathogenic Escherichia coli (APEC) ( 3 ). Syndromes associated with colibacillosis can vary, and include acute fatal septicemia, airsacculitis, pericarditis, perihepatitis, peritonitis, and lymphocytic depletion of the bursa and thymus. ( 4 ); salpingitis and cellulitis ( 5 ). In laying hens, peritonitis and salpingitis are prevalent, whereas disease in young chicks may include omphalitis (yolk sac infection) or swollen head syndrome ( 4 ). Previously, most APEC isolates were assigned to three main serogroups: O1, O2, and O₇₈; however, it has been demonstrated that there is a significant diversity in serogroups of APEC causing colibacillosis ( 6 ). Colibacillosis is one of the most prevalent and economically detrimental bacterial diseases of poultry worldwide ( 7 ), resulting in multimillion-dollar losses annually that affect numerous aspects of poultry production. The challenge of controlling colibacillosis is compounded by the pervasive antimicrobial resistance among APEC isolates ( 8 ), limitations on the use of antimicrobial agents in poultry, and the absence of vaccines that offer comprehensive protection against all types of APEC isolates associated with colibacillosis. There are reports on Escherichia coli autogenous vaccines, predominantly utilized in breeder flocks; however, evidence on the efficacy of such vaccines in terms of mitigating E. coli infections is scarce. The present study was therefore undertaken to evaluate the efficacy of humoral immunity response in layer chickens vaccinated with an Escherichia coli vaccine developed at the Shiraz Razi Institute.

2. Materials and Methods

2.1. Animals

A total of sixty unvaccinated layer chickens (Hy-line) of mixed sex were obtained on the day of hatching from a commercial hatchery of the Razi Vaccine and Serum Research Institute (Shiraz branch). These birds were then kept in a controlled area with free access to food and water at the poultry department of the Razi Vaccine and Serum Research Institute (Shiraz branch).

2.2. Isolation, Identification, and Serotyping

Poultry pathogenic Escherichia coli serotype O₇₈ was obtained from the microbiology department of the Razi Vaccine and Serum Research Institute (Shiraz branch), this bacterium selected for this study was isolated in the laboratory from the hearts and livers of 4–8-day-old broiler chickens suffering from colibacillosis infection with perihepatitis and pericarditis. For isolation of E. coli, tryptic soy broth (Merck, Germany), MacConkey agar (MCA) and Eosin-methylene blue (EMB) agar were used as enrichment, differential and selective medium respectively. The enrichment, the MCA, and EMB agar were incubated at 37°C for 24 hr. The smooth, moist colonies having metallic sheen on EMB agar were randomly sub-cultured. The isolates were identified on the basis of their cultural, morphological and biochemical characteristics ( 9 ). Identification of Escherichia coli O₇₈ was done by polymerase chain reaction (PCR) in experimentally infected specimens ( 10 ). The PCR amplifications were conducted in a 25 μL reaction volume containing 12.5 μL of Master Mix 2X AMPLIQON (Denmark), 1.5 U Taq polymerase, 1.5 Mm MgCl₂ ,1 μL of each primer, PCR buffer, and RNase-free water. To confirm Escherichia coli strain O₇₈ from PCR test using forward and reverse primers OG78 related to wzx gene was used based on the presence of 992 bp fragment (Table 1; Figure 1). Polymerase chain reaction was done in an Eppendorf thermocycler (Germany) during 30 cycles with denaturing temperature of 94°C for one minute, annealing temperature of 55°C for 40 seconds, and an extension temperature of 72°C for 1 minute was done. The polymerase chain reaction product was electrophoresed on a 1% agarose gel (Figure 1). Poultry pathogenic Escherichia coli serotype O₇₈ was obtained from the microbiology department of the Razi Vaccine and Serum Research Institute (Shiraz branch). The bacterium was isolated in the laboratory from the hearts and livers of 4–8-day-old broiler chickens suffering from colibacillosis infection with perihepatitis and pericarditis. The isolation of E. coli was performed using tryptic soy broth (Merck, Germany), MacConkey agar (MCA) and eosin-methylene blue (EMB) agar as enrichment, differential and selective media, respectively. These were incubated at 37°C for 24 hours. Smooth, moist colonies with a metallic sheen on EMB agar were then randomly sub-cultured. The isolates were identified on the basis of their cultural, morphological and biochemical characteristics ( 9 ). The identification of Escherichia coli O₇₈ was done by polymerase chain reaction (PCR) in experimentally infected specimens ( 10 ). The PCR amplifications were conducted in a 25 μL reaction volume containing 12.5 μL of Master Mix 2X AMPLIQON (Denmark), 1.5 U Taq polymerase, 1.5 Mm MgCl2, 1 μL of each primer, PCR buffer, and RNase-free water. The confirmation of the Escherichia coli strain O₇₈ from the PCR test using forward and reverse primers OG78 related to the wzx gene was based on the presence of a 992 bp fragment (Table 1 & Figure 1). The polymerase chain reaction (PCR) was performed in an Eppendorf thermocycler (Germany) for 30 cycles, with a denaturing temperature of 94°C for one minute, an annealing temperature of 55°C for 40 seconds, and an extension temperature of 72°C for one minute.The polymerase chain reaction product was then subjected to electrophoresis on a 1% agarose gel (Figure 1).

Figure 1. Molecular PCR test to confirm Escherichia coli O₇₈.

2.3. Antigens

The PCR confirmed isolate was proliferated in tryptic soy broth (Merck, Germany) (24 h/37°C). After incubation, cell suspension was inactivated with 0.3% formaldehyde at 37°C for 24 h. The cells were separated through centrifuging (5,000 g/20 min), and the pellet was resuspended in phosphate-buffered saline (PBS, pH 7.2). A suspension was adjusted to the turbidity standard 2 × 10⁹ bacteria / ml. The PCR-confirmed isolate was then propagated in tryptic soy broth (Merck, Germany) at 37°C for a duration of 24 hours. Following this incubation period, the cell suspension was inactivated using 0.3% formaldehyde at 37°C for a further 24 hours. Subsequently, the cells were separated through a process of centrifugation (5,000 g/20 minutes), after which the pellet was resuspended in phosphate-buffered saline (PBS, pH 7.2). The suspension was then adjusted to a turbidity standard of 2 ×10⁹ bacteria/ml.

2.4. Adjuvants and Vaccines

The production of oil-adjuvanted vaccines involved the preparation of standard W/O emulsions, with Montanide^TM ISA 70 being utilized as an example. These emulsions were subjected to gentle agitation in a mixer at room temperature. The subsequent step involved the combination of the aqueous phase, with a ratio of 70:30 (w/w, adjuvant: antigen or PBS), as recommended by the adjuvant manufacturer, Seppic (France).

2.5. Immunization of Chickens

A total of 60 chickens were randomly allocated to six treatment groups (ten birds each), namely the control group, and the groups designated T1, T2, T3, T4, and T5. The birds in the control group were not given E. coli vaccines; those in the groups T1, T2, T3, T4, and T5 were vaccinated at 28 days of age with 0.2 ml of the vaccine (8 ×10⁶, 16 ×10⁶, 33 ×10⁶, respectively). 06, 66 ×10⁶, 133 ×10⁶ cfu/ml per dose of E. coli O₇₈, respectively. The vaccine was formulated with formalin-inactivated, mineral-oil-adjuvanted E. coli (serotype O₇₈) vaccine subcutaneously.

2.6. Serum Titer of Anti- E. coli O78 Antibody

In this study, a total of 60 chickens were randomly selected for blood sample collection from the brachial vein prior to immunization. Subsequent to this, additional blood samples were collected from 10 chickens per group on a weekly basis, continuing for a period of 10 weeks following immunization. The separated sera were then stored at -20°C until analysis of the antibody responses against E. coli O₇₈ using an indirect enzyme-linked immunosorbent assay (ELISA).

2.7. Serology

The antigen response following challenge with E. coli O₇₈ was defined by Enzyme-Linked Immunosorbent Assay (ELISA). As previously mentioned, the samples were subjected to centrifugation at 3500×g for 10 minutes, after which the serum fraction was transferred to separate tubes and stored at -20 °C until ELISA evaluation of antibody content. The ELISA assay was performed on 96-well plates, which were coated overnight at 4 °C with 0.5 μg of whole cell sonicates of E. coli O₇₈. The sonicates were then diluted with carbonate-bicarbonate buffer (pH 9.6). Subsequent to this, each well was washed by adding 300 μL of washing buffer (PBS + 0.05% Tween 20). Following this, 200 μL of bovine serum albumin (BSA) was added as a blocking solution and the plates were left to stand at room temperature for 2 hours. After this time, the blocking solution was removed and the plates were washed. Following this, 100 μL of serum samples were added to each well. After incubation and washing, 100 μL polyclonal goat anti-chicken IgG diluted 1:4000 in diluting buffer were added to each well and the plates incubated for 1 h at 37 °C. Then, the plates washed. To ascertain the binding, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added to each well and incubated for 15 minutes. The reaction was then halted by the addition of 100 μL 1 M H₂SO₄, and the optical density was measured at 450 nm using a spectrophotometer (BioTek Instruments).

3. Results

The results of the serological tests are displayed in Table 2. The data demonstrated a significant increase in IgG antibody titers in the immunized birds in comparison to the unimmunized control group (P <0.05; Figure 2). The E. coli vaccine, which was developed in Shiraz at the Razi Institute, elicited higher IgG titers in most weeks (Figures 3 and 4). No differences in antibody titers against Escherichia coli were found between the experimental and control groups at weeks 5 to 10 (Figures 4 and 5). The mean OD titer of sera from the vaccinated groups was generally higher than that of the control groups (Figure 2). However, no differences were found between the T5 and T1 groups, but significant differences were found in the T5 group compared to the corresponding values for the T2, T3, and T4 groups.

Figure 2. Mean titre of OD of sera of laying chickens following vaccination with Escherichia coli O₇₈ which produced in Shiraz, Razi Institute. T₁, T₂, T₃, T₄, and T₅ groups were vaccinated at 28 days of age with 0.2 ml (8 × 10⁶, 16 × 10⁶, 33 × 10⁶, 66 × 10⁰6, and 133 × 10⁶ cfu/ml) per dose of E. coli O₇₈ respectively. Means with different letters differ significantly (P ≤ 0.05).

Figure 3. Effect of treat × time (wk) interaction on antibody titers against E. Coli (SP%) in Hyline selected laying chickens at different weeks post-immunization. T₁, T₂, T₃, T₄, and T₅ groups were vaccinated at 28 days of age with 0.2 ml (8 × 10⁶, 16 × 10⁶, 33 × 10⁶, 66 ×10⁶, and 133 × 10⁶ cfu/ml) per dose of E. coli O₇₈ respectively. ^a,bWithin each week, least squares means with different letters differ significantly (P ≤ 0.05).

Figure 4. Effect of treat × time (wk) interaction on antibody titers against E. Coli (SP%) in Hyline selected laying chickens at different weeks post-immunization. T₁, T₂, T₃, T₄, and T₅ groups were vaccinated at 28 days of age with 0.2 ml (8 × 10⁶, 16 × 10⁶, 33 × 10⁶, 66 × 10⁶, and 133 × 10⁶ cfu/ml) per dose of E. coli O₇₈ respectively. ^a,bWithin each week, least squares means with different letters differ significantly (P ≤ 0.05). No differences were found at wk 5 and 6.

Figure 5. Effect of treat × time (wk) interaction on antibody titers against E. Coli (SP%) in Hyline selected laying chickens at different weeks post-immunization. T₁, T₂, T₃, T₄, and T₅ groups were vaccinated at 28 days of age with 0.2 ml (8 × 10⁶, 16 × 10⁶, 33 × 10⁶, 66 × 10⁶, and 133 × 10⁶ cfu/ml) per dose of E. coli O₇₈ respectively. No differences were found at wk 7, 8, 9, and 10.

4. Discussion

As before mentioned, colibacillosis is an economically important for the avian industry, which causes multimillion-dollar losses annually ( 11 ). Preparing effectual colibacillosis control measures highly favorable. Colibacillosis control mostly concentrates on management methods made biosecurity plan for reduce preparing conditions among production birds, such as mycoplasma or viral infections ( 11 ). Although, management methods that have reduced colibacillosis in the past may not be as efficient in the future. Moreover, use of antimicrobial factors in animal production is being given close investigation at this time with restriction being placed on the use of certain therapeutic factors in avian production ( 12 ). Finally, control of avian colibacillosis using vaccines in specified conditions may demonstrate favorable. Up to the present time, vaccines formulated to impede avian colibacillosis have been faced with mixed results. Vaccines against APEC of different serogroups have been generated. Killed bacterial vaccines, including autogenous vaccines, sub-unit vaccines, and live-attenuated vaccines are in use for prevention of APEC ( 13 - 15 ). As previously stated, colibacillosis represents a significant economic burden on the avian industry, causing losses amounting to millions of dollars on an annual basis ( 11 ). The development of effective control measures for colibacillosis is of paramount importance. These measures primarily focus on the implementation of biosecurity plans aimed at mitigating risk factors such as mycoplasma or viral infections in production birds ( 11 ). However, it should be noted that the efficacy of management methods employed in the past to control colibacillosis may not be guaranteed in the future. Furthermore, the use of antimicrobial factors in animal production is currently under close investigation, with restrictions being placed on the use of certain therapeutic factors in avian production ( 12 ). Finally, the use of vaccines to control avian colibacillosis in specified conditions may prove to be advantageous. To date, vaccines formulated to impede avian colibacillosis have yielded equivocal outcomes. Vaccines against APEC of different serogroups have been generated, including killed bacterial vaccines, such as autogenous vaccines, sub-unit vaccines, and live-attenuated vaccines, which are in use for the prevention of APEC ( 13 - 15 ). A great number of these vaccines have only been effective against homologous challenge. The present report is to address the efficacy of humoral immunity response of killed oil adjuvant Escherichia coli vaccine in layer chicken against avian E. coli infection. The titer in IgG antibodies in the experimental groups were higher compared to the control group. Increased titer in IgG antibodies was more pronounced in T₅` birds which receiving the highest number of bacteria per mL. Śmiałek et al. ( 16 ) showed that the use of live, attenuated, aroA gene-deleted vaccine against colibacillosis cause a reduction in the amount of E. coli in the population of avian. These results are in agreement with findings of El-Mawgoud et al. ( 17 ), who showed that live E. coli spray vaccination of broiler chickens decreased the APEC colonization in the liver and heart of the birds after E. coli infection. Also Roland et al. ( 18 ) reported the use of live, attenuated, E. coli vaccine derived O₇₈ LPS, protected the white leghorn chicks against avian pathogenic E coli O₇₈ strain. In the present study killed Escherichia coli vaccine was used, because the production process of live vaccine requires a lyophilizer and the facility of live vaccine preparation was not available in Shiraz, Razi Institute. Killed E. coli vaccines, including autogenous vaccines, protect only against homologous challenge ( 5 , 19 ). A significant proportion of these vaccines have only demonstrated efficacy against homologous challenge (Smith et al., 2022). The present report aims to address the efficacy of humoral immunity response of killed oil adjuvant Escherichia coli vaccine in layer chicken against avian E. coli infection.The experimental groups exhibited higher titres of IgG antibodies in comparison to the control group, with the increase being more pronounced in T5 birds, which received the highest number of bacteria per mL. In a related study, Śmiałek et al. ( 16 ) demonstrated that the utilisation of a live, attenuated, aroA gene-deleted vaccine against colibacillosis resulted in a decline in the population of E. coli. These results align with the findings of El-Mawgoud et al. ( 17 ), who demonstrated that live E. coli spray vaccination of broiler chickens led to a reduction in APEC colonization in the liver and heart of the birds following E. coli infection.Additionally, Roland et al. ( 18 ) reported that the use of a live, attenuated E. coli vaccine derived from O₇₈ LPS provided protection to white leghorn chicks against the avian pathogenic E. coli O₇₈ strain. In the present study, a killed Escherichia coli vaccine was utilised due to the requirement of a lyophilizer for the production of a live vaccine, which was not available at the Shiraz Razi Institute. It is noteworthy that killed E. coli vaccines, including autogenous vaccines, offer protection exclusively against homologous challenges ( 5 , 19 ). Finally, the use of killed vaccines requires knowledge of the serotype(s) of E. coli, which are included in the substantial outbreaks. Unfortunately, vaccination with killed vaccines may stress the birds, and the adjuvants may induce local reactions ( 20 ). Sub-unit vaccines may provide an extensive protection against more serotypes of APEC. However, the disadvantage of stress to birds during vaccination, and side effects of adjuvants have also been recorded for the sub-unit vaccine ( 20 ). Vaccination of broiler parents by the inactivated subunit Nobilis® E. coli was found to reduce the number of sequence types of E. coli isolated from diseased broiler parents in the vaccinated flock compared to the control group, which shows a potential for sub-unit vaccine to make less the outbreak of specific clones of APEC ( 21 ). The live Poulvac® E. coli vaccine includes an aroA mutant of a strain of serotype O₇₈:K80 and ST23, but protection is not limited to this specific serotype and sequence type ( 22 ). Recent experimental studies have combined vaccination with live attenuated E. coli vaccine with autogenous vaccines and it seems possible to take a synergy of protection ( 23 ). The investigation of Kariyawasam et al. ( 24 ) revealed that collected IgY from eggs took from hens under different vaccination programs could cause passive maternal protection of day-old chicks when E. coli was used for challenge compared to the control groups, documenting that vaccination of parents may transfer the immunity to the chicks, under experimental conditions. Based on the results of this study the application of killed oil adjuvant Escherichia coli vaccine which produced in Shiraz, Razi Institute had greater efficacy in rising IgG titers in layer hens in comparison with unvaccinated group; however, to evoke immunological response, the second immunization is suggested four weeks after the first immunization. Finally, it is imperative to acknowledge the necessity of understanding the serotype(s) of E. coli implicated in substantial outbreaks, particularly in the context of the utilization of killed vaccines. It is noteworthy that vaccination with killed vaccines has the potential to exert stress on birds, and the adjuvants employed may elicit local reactions ( 20 ). Conversely, sub-unit vaccines offer the advantage of providing extensive protection against a broader spectrum of APEC serotypes. However, it should be noted that stress to birds during vaccination and side effects of adjuvants have also been recorded for the sub-unit vaccine ( 20 ). The vaccination of broiler parents with the inactivated subunit Nobilis® E. coli has been demonstrated to reduce the number of sequence types of E. coli isolated from diseased broiler parents in the vaccinated flock when compared to the control group. This finding suggests a potential for the sub-unit vaccine to mitigate the outbreak of specific clones of APEC ( 21 ). The live Poulvac® E. coli vaccine incorporates an aroA mutant of a strain of serotype O₇₈:K80 and ST23, but protection is not limited to these specific serotypes and sequence types ( 22 ). Recent experimental studies have combined vaccination with live attenuated E. coli vaccines with autogenous vaccines, and it seems possible to achieve a synergistic effect in terms of protection ( 23 ). The investigation by Kariyawasam et al. ( 24 ) revealed that IgY collected from eggs obtained from hens under different vaccination programs could elicit passive maternal protection in day-old chicks when E. coli was used for challenge, in comparison to the control groups. This suggests that vaccination of parents may transfer immunity to chicks under experimental conditions. The findings of this study indicate that the employment of a killed oil adjuvant Escherichia coli vaccine, which was produced in Shiraz at the Razi Institute, resulted in a higher level of IgG titers in layer hens when compared to the unvaccinated group. However, to elicit an immunological response, it is recommended that a second immunization be administered four weeks after the initial immunization.

Acknowledgment

The authors would like to express their gratitude to the Razi Vaccine and Serum Research Institute, Shiraz Branch.

Authors' Contribution

Study concept and design: A.S; M.H.H; F.S.

Acquisition of data: F.S; R.R; F.D; M.H; S.A; A.R.

Analysis and interpretation of data: F.S; M.H.

Drafting of the manuscript: F.S

Critical revision of the manuscript for important intellectual content: A.S; M.H; F.S.

Ethics

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

The present project was supported in part by the Razi Vaccine and Serum Research Institute, Shiraz Branch.

Data Availability

The data that underpin the findings of this study are available upon request from the corresponding author.

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