Evaluation of commercially available aroA delated gene E. coli O78 vaccine in commercial broiler chickens under Middle East simulating field conditions

The broiler industry in the Middle East (ME) faces many challenges related to bacterial infections, including M. gallisepticum, M. synoviae, E. coli, and other gram-negative bacteria, exacerbated by various errors in the brooding process. Antibiotics use in the first three days of life, such as Linco-Spectin 100 SP, tilmicosin, enrofloxacin, tylosin, colistin, and doxycycline, is the trend in the market to control such challenges. This study aimed to evaluate the efficacy of the newly introduced aroA E. coli vaccine (Poulvac E. coli) and its ability to reduce over-reliance on the heavy use of antibiotics in the ME. The study was conducted on 160 broiler chicks, divided into eight even groups. Each group was treated differently in terms of antibiotic therapy and ages at the time of Poulvac E. coli administration and the challenge of virulent avian pathogenic E. coli (APEC), serotype O78. Spray application of Poulvac E. coli at seven days of age plus Linco-Spectin 100 SP during the first three days provided the best results for zero mortality after challenge with APEC, while Poulvac E. coli at seven days with enrofloxacin during the early three days resulted in 10% mortality. Poulvac E. coli hatchery vaccination protected birds against mortality but reduced body weight gain compared to the 7-day group vaccinated with Linco-Spectin 100 SP during the first three days. Poulvac E. coli given on day one or day seven did not affect the immune response to concurrent respiratory viral vaccines and, in some cases, improved response. This study shows that Poulvac E. coli at seven days of age, together with Linco-Spectin 100 during the first three days, has produced the best results in terms of protection and performance in the ME high presence of avian pathogenic E. coli field challenge.

Poulvac E. coli live attenuated vaccine. Poulvac E. coli (Zoetis) is a live AroA gene that has been deleted from the Escherichia coli serotype O78 vaccine. The parent strain is virulent E. coli O78, isolated in the United Kingdom from the Central Veterinary Laboratories (CVL) in Weybridge in 1995. The initial vaccine lost its plasmid virulence and other storage virulence factors but retained the essential wall structure and metabolic profile of the O78 vaccine. Modification by deleting the aroA gene responsible for the biosynthesis of aromatic amino acids attenuated the Poulvac E. coli vaccine (Zoetis, Charles City Manufacturing site, USA), which was administered by coarse spray to these different experimental study groups either on day one or at 7 days of age.
Antibiotics. Two of the most commonly used antibiotics in the broiler industry were utilized and compared in this study, lincomycin-spectinomycin [Linco-Spectin 100 SP (Zoetis, Lot. 17043210A, Suzhou Manufacturing site, China)] and enrofloxacin [Baytril 10% Oral Solution (Bayer)] administered at 225 mg/kg and 10 mg/kg, respectively, during the first three days of drinking water life.
E. coli challenge strain. The E. coli O78 strain EC34195 was kindly offered by the Department of Microbiology of the University of Sadat City Faculty of Veterinary Medicine. The challenge was the intra-tracheal inoculation of 100 μl/bird (109 CFU per ml) at 28 days of age in those affected.
Experimental groups. The 160 experimental chicks were randomized and evenly assigned to different treatment groups, resulting in eight groups of twenty, consisting of two replicates. The design of the experimental study is shown in the following Table 1. Groups A1(LS-7dV-Ch) and A2(LS-7dV-NCh) were administered Clinical examination, necropsy, and histopathology. Seven days after exposure to E. coli, all chicks in the different experimental groups were observed for clinical signs of colibacillosis (ruffled feathers, rales, gasping, nasal discharge, and diarrhea). Dead birds were necropsied, the remaining birds out of 10 birds per treatment group were humanely killed, and post mortem (PM) tests were conducted under the established system 9 . Samples for histopathological examination originating from tracheas and liver of chicks were fixed in 10% neutral-buffered formalin, routinely processed, enclosed in paraffin, and cut and stained haematoxylin and eosin (HE) 10 . Characteristic lesions for colibacillosis infection have been reported, including cloudy air sacs with or without caseous exudate, peritonitis, perihepatitis, and pericarditis 11,12 . Blood samples. At 37 days of age, chicken blood was collected from the wing vein or by slaughtering and kept in sloped position at 37 °C for one hour then at 4 °C overnight. Sera were then separated by centrifugation at 3000 rpm/10 min and stored at − 20 °C until testing, which included the detection of H5N1-and NDVspecific antibodies using a hemagglutination inhibition (HI) assay, according to the World Organization of Animal Health (2008) 13 . Several clinical biochemistry parameters were also reported: total serum protein was determined by Biuret's Weichselbaum reaction 14 , bromcresol green was used to determine albumin at 628 nm 15 , blood urea was colorimetrically measured at 578 nm 16 , and creatinine has been calculated using the Jaffe reaction automatic kinetic process 17 .
Statistical analysis. Statistical analysis was performed using MedCalc Statistical Software 19.5.3. Average body weight and mortality were based on the Chi-square test analysis 18 . However, mean PM lesion was performed based on the Kruskal-Wallis test 19 , the serum biochemical parameters for non-challenged groups and the groups at issue were significantly different across groups.

Results
Clinical signs. Clinical signs observed following the E. coli challenge included depression, ruffled feathers, and mild diarrhea, with more severe, especially enteric, symptoms seen three days after the challenge. Nonchallenged groups remained healthy and asymptomatic.
Average body weight. The average body weight was evaluated and recorded at 23, 30, and 35 days of age, with lower final weights reached in the groups challenged with E. coli O78 strain. For 35 days in groups A1, B1, C1 and D1 (E. coli-challenged), body weights were 2.14 kg, 2.18 kg, 1.94 kg, and 1.95 kg, respectively, while for groups A2, B2, C2, and D2 (non-challenged), body weights were 2.34 kg, 2.42 kg, 2.4 kg, and 2.1 kg, respectively, as shown in Table 2. Where two-tailed probabilities (P) equal to 0.0323 and the Chi-square normal distribution test showed accepted normality for Chi-square equal to 0.934 and P equal to 0.3339 with Degrees of Freedom (DF) equal to 1 as shown in Table 3.
Mortality and post-mortem lesions. Mortality was observed and recorded for one week after E. coli O78 is a challenge. Mortality percentages in groups A1, B1, C1, and D1 (E. coli-challenged) were 0%, 10%, 0%, and 10%, respectively, while mortality remained 0 percent in groups A2, B2, C2, and D4 (non-challenged) as shown in Table 2. Table 4 shows that the result of mortality is not significant as Chi-squared equal to 8 and Degree of Freedom (DF) equal to 7 with P-Value equal to 0.3326. www.nature.com/scientificreports/ PM lesions were measured by cloudiness, turbidity, or deposition of the fibrin membrane to the liver, heart, and air sacs. The average PM lesion score for groups A1, B1, C1, and D1 (E. coli-challenged) was 2.6, 2.2, 2.5, and 3, respectively ( Table 2). Table 5 showed the nonparametric Kruskal-Wallis test. The p-value equal to 0.391625 greater than 0.05 indicates that the average PM lesion does not provide any reason to conclude that the distributions are different.

Serological evaluation of antibody titers for AI H5N1 and NDV by hemagglutination inhibition (HI) assay.
Due to limited human resources, ten birds per treatment group have been retained for blood work. The antibody titers for the avian influenza H5N1 virus were evaluated at 21, 27, and 37 days of age and found to be approximately identical between groups. At 37 days, the HI titer (log2) for E. coli-challenged groups A1, B1, C1, and D1 was 6, 5.2, 5.7, 5.5, respectively, while the titer for non-challenged groups A2, B2, C2, and D4 Table 2. Average body weight, mortality, and PM lesion scores. Means with different letters (a, b) are significantly different at P-value ≤ 0.05. *A1: LS-7dV-Ch, A2: LS-7dV-NCh, B1: En-7dV-Ch, B2: En-7dV-NCh, C1: 1dV-Ch, C2: 1dV-Ch, D1: En-Nv-Ch, D2: En-Nv-NCh. **No PM lesions have been observed.   www.nature.com/scientificreports/ was 6.5, 4.5, 5.7, and 6, respectively, as shown in Table 6. The antibody titers shown against the avian influenza H5N1 virus P-value of 0.368 and the Chi-square normal distribution test showed accepted normality for Chisquare equal to 0.277 and P equal 0.5986 with Degrees of Freedom (DF) equal to 1 (Table 7). Antibody titers for NDV virus were evaluated at 21, 27, and 37 days of age. At 37 days, the HI titer (log2) for E. coli-challenged groups A1, B1, C1, and D1 was 6.5, 6.2, 5.5, and 5.5, respectively, while the titer for nonchallenged groups A2, B2, C2, and D4 was 5, 5, 5.7 and 5.7, respectively, as shown in Table 6. Table 8 showed antibody titers for the NDV virus P-value of 0.042. The chi-square normal distribution test showed accepted normality for Chi-square equal to 0.115 and P-value equal to 0.7349 with Degrees of Freedom (DF) equal to 1.
Histopathological results. Trachea. Histopathological examination revealed that between the different unchallenged groups. Figure 1 shows a normal trachea with a normal mucosal layer formed by pseudostrati-    (Figs. 3, 4). Table 9 showed the severity index of histopathological changes in the broiler chicken trachea in all groups.
Liver. Histopathological changes in different groups showed mild congestion of the blood vessels, and hepatocytes exhibited mild to severe vacuolar degeneration. Group A1 (LS-7dV-Ch) offers a focal aggregation of histiocytes (Fig. 5), while groups B1 (En-7dV-Ch) & C1 (1dV-Ch) exhibit congestion in the blood sinusoid with thrombus formation and bile ductule proliferation (Figs. 6, 7). Group D1 (En-Nv-Ch) showed severe congestion with prominent thrombus formation and inflammatory cell aggregation. The severity index for each histopathological change in the liver of broiler chickens in all groups has been shown in Table 10.

Results of serum biochemical parameters.
Statistical analyses of the results of the total serum protein, albumin, urea, and creatinine E. coli-challenged and non-challenged groups are shown below in Tables 11, 12; www.nature.com/scientificreports/ Statistical analyses of the results of serum biochemistry revealed that mean values of total serum proteins, albumin, urea, and creatinine showed non-significant changes between non-challenged groups (A2, B2, C2, and D2) with only significant differences in total protein between samples collected from Groups A2& B2 and those collected from Groups C2& D2.
Statistical analysis of total protein, urea, and creatinine results revealed significant differences between different E. coli challenged groups; however, there were no significant differences between different groups for total albumin. In other words, the A1 group showed a significant increase in total protein compared to group D1. Meanwhile, groups B1 & D1 showed a significant increase in urea and creatinine than groups A1& C1.

Discussion
E. coli are ubiquitous bacteria found in all poultry intestines, and most strains may be considered non-pathogenic, part of the microbiome-related bacterial community.
However, some strains, including extra-intestinal avian pathogenic E. coli (APEC), can cause invasive infections outside the intestine, namely colibacillosis 1,2 , and are reported to cause significant economic losses to the poultry industry 20 . ME Poulvac E coli sprays application of day-old broilers has been reported to help reduce the mortality associated with co-infection by IBV variant 02 and APEC from 25% in the control group 10% 21 .  www.nature.com/scientificreports/ However, under ME field conditions and due to the low quality of day-old chicks, broiler chick medicines are common in the first three days of age to eliminate vertical or hatchery-transmitted bacterial pathogens. Simultaneously, in vitro assays have already shown that Poulvac E. coli is sensitive to Linco-Spectin 100 SP and Baytril 10%, commonly used in ME. Our objective was to measure any effect that common practice might have on the efficacy (in vivo) of Poulvac E. coli to best position the vaccine with two heavily used broiler-brooded antibiotics.
Chicks in experimental groups A1 (LS-7dV-Ch) and A2 (LS-7dV-NCh) were both administered Linco-Spectin 100 during the first three days of life and vaccinated with Poulvac E. coli on day 7.
Chicks in groups B1 (En-7dV-Ch) & B2 (En-7dV-NCh) were both given enrofloxacin for the first 3 days of life and vaccinated with Poulvac E. coli on day 7. Chicks in groups C1 (1dV-Ch) & C2 (1dV-NCh) were vaccinated with Poulvac E. coli on 1 day of age. Finally, chicks in groups D1 (En-Nv-Ch) and D2 (En-Nv-NCh) were administered enrofloxacin for the first three days of their life. Chicks in groups A1, B1, C1, and D1, were exposed to the virulent strain of E. coli O78 on day 28. All chicks in the different experimental groups were observed for clinical signs of colibacillosis, mortality, PM lesions, histopathological changes, serological evaluation of AIV H5N1 and NDV, and clinicopathological changes.
The results of this study showed that all vaccine treatments had significant reductions in the incidence and severity of airsacculitis-by far the most common lesion (groups A1, B1, and C1). However, there was a difference in E. coli protection between the different groups based on prior antibiotic prophylaxis. Out of the four groups in a challenge, birds in groups A1 (LS-7dV-Ch) and C1 (1dV-Ch) showed 100% protection against mortality, while birds in groups B1 (En-7dV-Ch) and D1 (En-Nv-Ch) showed 10% mortality. The efficacy of Poulvac E. coli given daily by coarse spray was not compromised by prior gentamicin or Naxcel injection, either in ovo or

Groups
Congestion Hemorrhage

Hyperplasia of lining epithelium
Inflammatory cells www.nature.com/scientificreports/ subQ. This suggests that these antibiotics' levels were insufficient to interfere with the interaction between the vaccine and the chick's immune system daily 8 .

Epithelial degeneration Mucous glands
In group C1 (1dV-Ch), despite birds' protection against mortality, the average body weights at day 35 were numerically lower than birds of group A1 (LS-7dV-Ch). For non-challenged groups, all vaccine treatments [groups A2 (LS-7dV-NCh), B2 (En-7dV-NCh), and C2 (1dV-NCh)] showed a higher average body weight at 35 days than non-vaccinated group D2 (En-Nv-NCh). The observed results were similar to previous study and to the recent field trial in Saudi Arabia where Linco-Spectin 100/Poulvac E. coli treated flocks showed superior average live body weight, dressed weight, FCR and EPI compared to their current antibiotics only treated flocks with 58 g, 47 g, 5 points and 10.53 points of differences, respectively 22,23 .
The effect of Poulvac E. coli on the immune response to concurrent AI H5 and NDV vaccinations was evaluated by the detection of antibody titers at different ages against each particular agent by the HI test. Results have shown that the use of Poulvac E. coli on day-old or day 7 with prior antibiotic prophylaxis in the first three days    www.nature.com/scientificreports/ does not affect the immune response to these vaccines. Also, there was an improvement in both viral agents' immune responses, particularly in group A1 (LS-7dV-Ch) 21,24 . The histopathological examination results showed a normal histological pattern of tracheas collected from birds of non-challenged groups with mild histopathological changes in the liver from the same birds. While the histopathological severity index varied between the different challenged groups, birds of group A1(LS-7dV-Ch) showed the mildest tracheal (mild focal thickening of the epithelium with submucosal lymphocytes nodules + 1) and liver lesions (focal aggregation of histiocytes in the liver), followed by birds in groups B1(En-7dV-Ch) & C1(1dV-Ch) (moderate lesions) and D1(En-Nv-Ch) (severe lesions), respectively.
These results suggest that treatments with the Poulvac E. coli vaccine significantly reduced the severity of lesions in both the trachea and liver compared to the non-vaccinated group D1(En-Nv-Ch) with a higher level of protection in group A1 (LS-7dV-Ch) 21,25 . The serum biochemical parameters were aligned with clinical signs, mortality, necropsy, and histopathology findings where group A1(LS-7dV-Ch) showed significant differences in total protein, urea, and creatinine compared to group D1 (En-Nv-Ch). Higher total protein, combined with a lack of difference in total albumin, indicates higher globulin levels in group A1 (LS-7dV-Ch) birds. Meanwhile, urea and creatinine levels in groups A1(LS-7dV-Ch) and C1(1dV-Ch) were significantly lower than in groups B1(En-7dV-Ch) and D1(En-Nv-Ch).

Conclusion
These results conclude the efficacy of Poulvac E. coli given at seven days old by coarse spray was not compromised by prior Linco-Spectin 100 medication at 1st 3 days in commercial broilers. On the other hand, the enrofloxacin level slightly interfered with this vaccine's interaction and the chick's immune system. The protection may not have been as complete as reflected in the 10% mortality. This study proves that Poulvac E. coli at seven days of age, together with Linco-Spectin 100 during the first 3 days, gave the best results in terms of protection and performance in the ME that has a high presence of avian pathogenic E. coli field challenge.