Vaccinating parent flocks against colibacillosis reduces broiler mortality - A retrospective observational study from 2016 to 2019 in Finland.

Colibacillosis is one of the most important infectious diseases in modern poultry production. The complex nature of colibacillosis has made it challenging to produce an effective vaccine. As a control measure for colibacillosis outbreak in Finland, a vaccination program with a commercial colibacillosis vaccine and later also an autogenous vaccine was started for parent flocks in 2017. In this retrospective observational study from years 2016-2019, we evaluated first week and total mortality of broiler flocks (n= 6969) originating from parents with different colibacillosis vaccination status. Broiler flocks were divided into three groups according to vaccination status of their parent flocks. First group were flocks from parents with no colibacillosis vaccines; second group was flocks from parents vaccinated with commercial vaccine only; and third group was flocks from parents with both commercial and autogenous vaccine. Bayesian modelling was used to predict posterior distributions of first week mortality and total mortality of the broiler flocks. Results of the modelling revealed that broiler flocks from unvaccinated parents had the highest mortality rates (mean first week mortality 1.40 % and mean total mortality 4.33 %, respectively) whereas flocks from parents with a combination of commercial and autogenous vaccinations had the lowest mortality rates (mean first week mortality 0,91 % and mean total mortality 3,14 %). The mortalities from broilers flocks from parents with only commercial vaccine fell in between these groups. Also, standard deviations of mortality rates were lower in broilers from parents with commercial or both vaccines. This demonstrates that in addition to lowering the mean mortality rates, the vaccinations made high mortality broiler flocks less common. Best performance was obtained when autogenous vaccine was combined to the commercial vaccine. The autogenous vaccine consists of the same type of Escherichia coli strain that was causing most colibacillosis cases during the study period in Finland. This study adds to the evidence of benefits of colibacillosis vaccines during outbreaks. It also demonstrates the importance of the knowledge of the types of APEC strains causing outbreaks to produce effective autogenous vaccines.

A systematic review on the role of biosecurity to prevent or control colibacillosis in broiler production.

This systematic review aimed at investigating the role that biosecurity can have in preventing or controlling colibacillosis in broiler production. Primary studies with natural or experimental exposure to avian pathogenic Escherichia coli, evaluating any biosecurity measure to prevent or control colibacillosis in broiler chickens with at least one of the following outcomes: feed conversion ratio (FCR), condemnations at slaughter, and mortality due to colibacillosis, were included. A systematic search was carried out in 4 databases according to the Cochrane handbook and reported following the PRISMA 2020 directions. Studies (n = 3,886) were screened in a 2-phase process and data matching the inclusion criteria were extracted. Risk of bias assessment was performed. Four studies reporting biosecurity measures to prevent or control colibacillosis in broiler production were included. In all studies, only disinfection during either the pre-hatching period (n = 3) or the post-hatching period (n = 1) was evaluated as biosecurity measure in broiler production, as well as its effect on FCR (n = 2) and mortality (n = 4) due to colibacillosis. No studies with effects on condemnations at slaughter were found. Due to the heterogeneity of studies in regard to interventions and outcomes, meta-analysis was not carried out. The limited findings of this systematic review do not provide a comprehensive evidence to statistically evaluate the efficacy of biosecurity to prevent or control colibacillosis in broiler production. The scarcity of evidence found suggests that further and deeper investigations on the topic are needed, considering the variety of interventions related to biosecurity.

Swine Colibacillosis: Analysis of the Gut Bacterial Microbiome.

This study aimed to evaluate the disruption of the swine gut microbiota and histopathological changes caused by infection with enterotoxigenic E. coli. Fecal samples were collected from piglets suffering from diarrhea post-recovery and healthy animals. Intestinal tissues were collected for histopathological changes. The results revealed histopathological changes mainly in the ileum of the infected animals compared to those in the ileum of the control and recovered animals. The operational taxonomic units (OTUs) revealed that the E. coli diarrheal group exhibited the highest bacterial richness. Principal coordinate analysis (PCoA) corroborated the presence of dysbiosis in the gut microbiota following E. coli-induced diarrhea. While the normal control and infected groups displayed slight clustering, the recovery group formed a distinct cluster with a distinct flora. Bacteroidetes, Firmicutes, and Fusobacteria were the dominant phyla in both the healthy and recovered piglets and in the diarrheal group. LEfSe and the associated LDA score analysis revealed that the recovered group exhibited dominance of the phyla Euryarchaeota and Bacteroidota, while groups N and I showed dominance of the phyla Firmicutes and Fusobacteriota, respectively. The LDA scores highlighted a significant expression of the Muribaculacea family in group R. The obtained findings will help in understanding the microbiome during swine colibacillosis, which will support control of the outbreaks.

Assessment of antibacterial activity, modes of action, and synergistic effects of Origanum vulgare hydroethanolic extract with antibiotics against avian pathogenic Escherichia coli.

This study evaluates the antibacterial effectiveness of Origanum vulgare hydroethanolic extract, both independently and in combination with antibiotics, against Escherichia coli strains associated with avian colibacillosis-a significant concern for the poultry industry due to the rise of antibiotic-resistant E. coli. The urgent demand for new treatments is addressed by analyzing the extract's phytochemical makeup via High-Performance Liquid Chromatography (HPLC), which identified sixteen phenolic compounds. Antibacterial activity was determined through agar diffusion and the measurement of minimum inhibitory and bactericidal concentrations (MIC and MBC), showing moderate efficacy (MIC: 3.9 to 7.8 mg/mL, MBC: 31.2 to 62.4 mg/mL). Combining the extract with antibiotics like ampicillin and tetracycline amplified antibacterial activity, indicating a synergistic effect and highlighting the importance of combinatory treatments against resistant strains. Further analysis revealed the extract's mechanisms of action include disrupting bacterial cell membrane integrity and inhibiting ATPase/H+ proton pumps, essential for bacterial survival. Moreover, the extract effectively inhibited and eradicated biofilms, crucial for preventing bacterial colonization. Regarding cytotoxicity, the extract showed no hemolytic effect at 1 to 9 mg/mL concentrations. These results suggest Origanum vulgare extract, particularly when used with antibiotics, offers a promising strategy for managing avian colibacillosis, providing both direct antibacterial benefits and moderating antibiotic resistance, thus potentially reducing the economic impact of the disease on the poultry industry.

Paeoniflorin protects chicken against APEC-induced acute lung injury by affecting the endocannabinoid system and inhibiting the PI3K/AKT and NF-κB signaling pathways.

Avian pathogenic Escherichia coli (APEC) is the causative agent of chicken colibacillosis. Paeoniflorin, a natural ingredient extracted from Paeonia lactiflora, has a variety of pharmacological effects including anti-inflammatory and immunomodulatory. However, its effects and mechanism in APEC-induced acute lung injury (ALI) in chicken is not clear. The aim of this study was to investigate the protective effect of paeoniflorin on APEC-induced ALI and its possible mechanism. Paeoniflorin (25, 50, and 100 mg/kg) was administered by gavage for 5 d starting at 9 d of age and the chicken were infected with APEC by intraperitoneal injection at 12 d of age. The tissues were collected after APEC infection for 36 h for analysis. The results showed that paeoniflorin significantly alleviated the symptoms, increased the survival rate and body weight gain of APEC-infected chicken, and improved the histopathological damages, and reduced APEC loads in lung tissues. In addition, paeoniflorin restored the gene expression of ZO-1, Occludin and Claudin-3 during APEC infection. Moreover, paeoniflorin pretreatment significantly affected the endocannabinoid system (ECs) by increasing DAGL, decreasing MAGL, increasing secretion of 2-AG. Then, paeoniflorin significantly decreased the secretion of IL-1ß, IL-6 and TNF-α in lung tissues, and decreased the mRNA expression of CXCL8, CXCL12, CCL1, CCL5, and CCL17. In addition, paeoniflorin significantly reduced the phosphorylation levels of PI3K, AKT, P65, and IκB. In summary, we found that paeoniflorin inhibited APEC-induced ALI, and its mechanism may be through affecting ECs and inhibiting the activation of PI3K/AKT and NF-κB signaling pathways, which provides a new idea for the prevention and treatment of chicken colibacillosis.

High frequency of multidrug-resistant Escherichia coli from cattle in the Cerrado and Pantanal biomes of Brazil.

The indiscriminate use of antimicrobials has led to the emergence of resistant bacteria, especially pathogenic strains of Escherichia coli, which are associated with diseases in animals and humans. The aim of the present study was to characterize E. coli isolates in calves with regards to the presence of virulence genes and investigate the resistance of the isolates to different antimicrobials. Between 2021 and 2023, 456 fecal samples were collected from calves in the Pantanal and Cerrado biomes of the state of Mato Grosso do Sul, Brazil. All samples were subjected to microbiological analysis and disc diffusion antibiogram testing. The polymerase chain reaction method was used to detect virulence genes. Bacterial growth was found in 451 of the 456 samples and biochemically identified as Escherichia coli. All 451 isolates (100 %) exhibited some phenotypic resistance to antimicrobials and 67.62 % exhibited multidrug resistance. The frequency of multidrug-resistant isolates in the Cerrado biome was significantly higher than that in the Pantanal biome (p = 0.0001). In the Cerrado, the most common pathotype was Shiga toxin-producing Escherichia coli (STEC) (28 %), followed by toxigenic Escherichia coli (ETEC) (11 %), enterohemorrhagic Escherichia coli (EHEC) (8 %) and enteropathogenic Escherichia coli (EPEC) (2 %). In most cases, the concomitant occurrence of pathotypes was more common, the most frequent of which were ETEC + STEC (33 %), ETEC + EHEC (15 %) and ETEC + EPEC (3 %). The STEC pathotype (30 %) was also found more frequently in the Pantanal, followed by EHEC (12 %), ETEC (9 %) and EPEC (6 %). The STEC pathotype had a significantly higher frequency of multidrug resistance (p = 0.0486) compared to the other pathotypes identified. The frequency of resistance was lower in strains from the Pantanal biome compared to those from the Cerrado biome. Although some factors are discussed in this paper, it is necessary to clarify the reasons for this difference and the possible impacts of these findings on both animal and human health in the region.

CATH-2-derived antimicrobial peptide inhibits multidrug-resistant Escherichia coli infection in chickens.

Avian colibacillosis (AC), caused by infection with Escherichia coli (E. coli), is a major threat to poultry health, food safety and public health, and results in high mortality and significant economic losses. Currently, new drugs are urgently needed to replace antibiotics due to the continuous emergence and increasing resistance of multidrug-resistant (MDR) strains of E. coli caused by the irrational use of antibiotics in agriculture and animal husbandry. In recent years, antimicrobial peptides (AMPs), which uniquely evolved to protect the host, have emerged as a leading alternative to antibiotics in clinical settings. CATH-2, a member of the antimicrobial cathelicidin peptide family, has been reported to have antibacterial activity. To enhance the antimicrobial potency and reduce the adverse effects on animals, we designed five novel AMPs, named C2-1, C2-2, C2-3, C2-4 and C2-5, based on chicken CATH-2, the secondary structures of these AMPs were consistently α-helical and had an altered net charge and hydrophobicity compared to those of the CATH-2 (1-15) sequences. Subsequently, the antimicrobial activities of CATH-2 (1-15) and five designed peptides against MDR E. coli were evaluated in vitro. Specifically, C2-2 showed excellent antimicrobial activity against either the ATCC standard strain or veterinary clinical isolates of MDR E. coli, with concentrations ranging from 2-8 µg/mL. Furthermore, C2-2 maintained its strong antibacterial efficacy under high temperature and saline conditions, demonstrating significant stability. Similarly, C2-2 retained a high level of safety with no significant hemolytic activity on chicken mature red blood cells or cytotoxicity on chicken kidney cells over the concentration range of 0-64 µg/mL. Moreover, the administration of C2-2 improved the survival rate and reduced the bacterial load in the heart, liver and spleen during MDR E. coli infection in chickens. Additionally, pathological damage to the heart, liver and intestine was prevented when MDR E. coli infected chickens were treated with C2-2. Together, our study showed that C2-2 may be a promising novel therapeutic agent for the treatment of MDR E. coli infections and AC.

In vitro evaluation of two novel Escherichia bacteriophages against multiple drug resistant avian pathogenic Escherichia coli.

BACKGROUND: In recent years, there has been a growing interest in phage therapy as an effective therapeutic tool against colibacillosis caused by avian pathogenic Escherichia coli (APEC) which resulted from the increasing number of multidrug resistant (MDR) APEC strains. METHODS: In the present study, we reported the characterization of a new lytic bacteriophage (Escherichia phage AG- MK-2022. Basu) isolated from poultry slaughterhouse wastewater. In addition, the in vitro bacteriolytic activity of the newly isolated phage (Escherichia phage AG- MK-2022. Basu) and the Escherichia phage VaT-2019a isolate PE17 (GenBank: MK353636.1) were assessed against MDR- APEC strains (n = 100) isolated from broiler chickens with clinical signs of colibacillosis. RESULTS: Escherichia phage AG- MK-2022. Basu belongs to the Myoviridae family and exhibits a broad host range. Furthermore, the phage showed stability under a wide range of temperatures, pH values and different concentrations of NaCl. Genome analysis of the Escherichia phage AG- MK-2022. Basu revealed that the phage possesses no antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and any E. coli virulence associated genes. In vitro bacterial challenge tests demonstrated that two phages, the Escherichia phage VaT-2019a isolate PE17 and the Escherichia phage AG- MK-2022. Basu exhibited high bactericidal activity against APEC strains and lysed 95% of the tested APEC strains. CONCLUSIONS: The current study findings indicate that both phages could be suggested as safe biocontrol agents and alternatives to antibiotics for controlling MDR-APEC strains isolated from broilers.

Virulence-associated genes in faecal and clinical Escherichia coli isolates cultured from broiler chickens in Australia.

A healthy chicken's intestinal flora harbours a rich reservoir of Escherichia coli as part of the commensal microbiota. However, some strains, known as avian pathogenic E. coli (APEC), carry specific virulence genes (VGs) that enable them to invade and cause extraintestinal infections such as avian colibacillosis. Although several VG combinations have been identified, the pathogenic mechanisms associated with APEC are ill-defined. The current study screened a subset of 88 E. coli isolates selected from 237 pre-existing isolates obtained from commercial poultry flocks in Australia. The 88 isolates were selected based on their enterobacterial repetitive intergenic consensus (ERIC) and antimicrobial resistance (AMR) profiles and included 29 E. coli isolates cultured from chickens with colibacillosis (referred to as clinical E. coli or CEC) and 59 faecal E. coli (FEC) isolates cultured from clinically healthy chickens. The isolates were screened for the presence of 35 previously reported VGs. Of these, 34 were identified, with iucA not being detected. VGs focG, hlyA and sfa/foc were only detected in FEC isolates. Eight VGs had a prevalence of 90% or above in the CEC isolates. Specifically, astA (100%); feoB (96.6%); iutA, iss, ompT, iroN and hlyF (all 93.1%); and vat (89.7%). The prevalence of these were significantly lower in FEC isolates (astA 79.7%, feoB 77.9%, iutA 52.5%, iss 45.8%, ompT 50.9%, iroN 37.3%, hlyF 50.9% and vat 42.4%). The odds ratios that each of these eight VGs were more likely to be associated with CEC than FEC ranged from 7.8 to 21.9. These eight VGs may be used to better define APEC and diagnostically detect APEC in Australia. Further investigations are needed to identify the roles of these VGs in pathogenicity.

Antimicrobial resistance of avian pathogenic Escherichia coli isolated from broiler, layer, and breeder chickens.

Background and Aim: Antimicrobials are extensively used in poultry production for growth promotion as well as for the treatment and control of diseases, including avian pathogenic Escherichia coli (APEC). Poor selection, overuse, and misuse of antimicrobial agents may promote the emergence and dissemination of antimicrobial resistance (AMR) in APEC. This study aimed to assess antimicrobial susceptibility patterns and detect antibiotic resistance genes (ARGs) in APEC isolated from clinical cases of colibacillosis in commercial broiler, layer, and breeder chickens. Materials and Methods: A total of 487 APEC were isolated from 539 across 300 poultry farms in various regions of Nepal. Antimicrobial susceptibility patterns was determined using the Kirby-Bauer disk diffusion and broth microdilution methods. The index of AMR, such as multiple antibiotic resistance (MAR) index, resistance score (R-score), and multidrug resistance (MDR) profile, were determined. Polymerase chain reaction was employed to detect multiple ARGs and correlations between phenotypic and genotypic resistance were analyzed. Results: The prevalence of APEC was 91% (487/539). All of these isolates were found resistant to at least one antimicrobial agent, and 41.7% of the isolates were resistant against 8-9 different antimicrobials. The antibiogram of APEC isolates overall showed the highest resistance against ampicillin (99.4%), whereas the highest intermediate resistance was observed in enrofloxacin (92%). The MAR index and R-score showed significant differences between broiler and layers, as well as between broiler breeder and layers. The number of isolates that were resistant to at least one agent in three or more antimicrobial categories tested was 446 (91.6%) and were classified as MDR-positive isolates. The ARGs were identified in 439 (90.1%) APEC isolates, including the most detected mobilized colistin resistance (mcr1) which was detected in the highest (52.6%) isolates. Overall, resistance gene of beta-lactam (blaTEM), mcr1, resistance gene of sulphonamide (sul1) and resistance gene of tetracycline (tetB) (in broiler), were detected in significantly higher than other tested genes (p < 0.001). When examining the pair-wise correlations, a significant phenotype-phenotype correlation (p < 0.001) was observed between levofloxacin and ciprofloxacin, chloramphenicol and tetracycline with doxycycline. Similarly, a significant phenotype-genotype correlation (p < 0.001) was observed between chloramphenicol and the tetB, and colistin with blaTEM and resistance gene of quinolone (qnrA). Conclusion: In this study, the current state of APEC AMR in commercial chickens is revealed for the first time in Nepal. We deciphered the complex nature of AMR in APEC populations. This information of molecular surveillance is useful to combat AMR in APEC and to contribute to manage APEC associated diseases and develop policies and guidelines to enhance the commercial chicken production.

Hepatoprotective and cardioprotective effect of Artemisia nilagirica leaf extract on E. coli challenged broiler chicken.

Artemisia nilagirica is an important medicinal plant found to exhibit several medicinal properties but the use of its leaves for combating E. coli infection has not been scientifically validated in poultry. The present study was conducted to evaluate the protective effects of methanol leaf extract of A. nilagirica (ANE) on E. coli challenged broiler chickens. Three hundred and thirty, day-old broiler chickens, were divided into 6 groups of 55 each, with group EX infected intraperitoneally (I/P) with LD50 dose of 1 × 107 cfu/ml of E. coli; group(s) EA1, EA2 and EA3 infected I/P with 1 × 107 cfu/ml of E. coli and supplemented with ANE @ 0.5, 1.0 and 2.0 g/L of drinking water, respectively; group AX were only given ANE @ 2.0 g/L in the drinking water. ANE treatment was started from day 4 and was continuously given in the drinking water up to day 21. E. coli infection was given to the birds on day 7 of their age. The effect of the plant extract was evaluated on the basis of gross, microscopic and ultrastructural alterations in E. coli challenged broiler chickens. The extract of A. nilagirica was found to show antibacterial, cardioprotective and hepatoprotective properties in a dose-dependent manner on the basis of gross and microscopic examination. The methanol extract of A. nilagirica leaves revealed no toxic effect on the hepatocytes on ultrastructural evaluation. This study demonstrates the antimicrobial, hepatoprotective and cardioprotective activities of ANE in broiler chickens infected with E. coli organism.

Evaluation of virulence factors in clinical isolates of pathogenic E. coli in avian samples in Caloto, Colombia

Avian pathogenic E. coli (APEC), produces an extraintestinal infection in chickens, turkeys, and other types of birds, called colibacillosis, which is considered one of the main causes of economic losses due to morbidity, mortality, and discard of poultry carcasses. The objective of the present study was to characterize the genetic profile of the virulence factors of different isolates of avian E. coli in Caloto, Cauca, Colombia. Materials and methods: E. coli was isolated and identified by biochemical tests, from 47 clinical isolates. Subsequently, the DNA was extracted using Chelex. Three multiplex PCRs were designed to amplify 13 virulence factors (iroN, hlyF, iss, iutA, frz, vat, sitA, KpsM, sitD, fimH, pstB, sopB, and uvrY), using primers previously reported for each. At the end, the amplification products were verified on agarose gels. Each isolate was classified according to the number of virulence factors: group A (between 10 and 13), group B (between 5 and 9), and group C (4 or less). Discussion and Conclusions: we were able to identify the presence of a group of virulence factors in clinical isolates of APEC, which allows us to demonstrate that both the frequency and the profile of virulence factors in the isolated strains showed a different profile than the reported by other authors. The virulence genes pstB and fimH were detected in all our samples, and the iss gene was the one with the lowest frequency. Finally, according to the number of virulence factors, the group A was the most frequent.

La E. coli patógena aviar (APEC), produce una infección extraintestinal en pollos, pavos y otros tipos de aves, denominada colibacilosis, la cual es considerada una de las principales causas de pérdidas económicas por morbilidad, mortalidad y descarte de canales de aves. El objetivo del presente estudio fue caracterizar el perfil genético de los factores de virulencia de diferentes aislamientos de E. coli aviar en Caloto, Cauca, Colombia. Materiales y métodos: E. coli se aisló e identificó mediante pruebas bioquímicas, a partir de 47 aislamientos clínicos. Posteriormente, el ADN se extrajo utilizando Chelex. Se diseñaron tres PCR multiplex para amplificar 13 factores de virulencia (iroN, hlyF, iss, iutA, frz, vat, sitA, KpsM, sitD, fimH, pstB, sopB y uvrY), utilizando primers informados previamente para cada uno. Al final, los productos de amplificación fueron verificados en geles de agarosa. Cada aislamiento se clasificó según el número de factores de virulencia: grupo A (entre 10 y 13), grupo B (entre 5 y 9) y grupo C (4 o menos). Discusión y Conclusiones: pudimos identificar la presencia de un grupo de factores de virulencia en los aislados clínicos de APEC, lo que nos permite demostrar que tanto la frecuencia como el perfil de los factores de virulencia en las cepas aisladas presentaron un perfil diferente al reportado por otros autores. Los genes de virulencia pstB y fimH se detectaron en todas nuestras muestras, siendo el gen iss el de menor frecuencia. Finalmente, según el número de factores de virulencia, el grupo A fue el más frecuente.

Avian Pathogenic Escherichia coli (APEC) in Broiler Breeders: An Overview.

Poultry meat is one of the major animal protein sources necessary to meet the global protein demand. Sustainability in broiler production is the key to achieving its continuous supply, and broiler breeders play a critical role in maintaining this sustainability by providing good quality chicks. Colibacillosis, the disease caused by avian pathogenic Escherichia coli (APEC), causes severe economic losses to the poultry industry globally. Moreover, APEC causes an additional burden among broiler breeders, such as a decrease in egg production and mortality among these birds. There is vertical transmission of APEC to the broiler chicks through eggs, resulting in increased first-week mortality and subsequent horizontal transmission at the hatchery. In this regard, the vertical transmission of antibiotic resistance genes is another concern that needs attention. Controlling several diseases in broiler breeders would possibly reduce the first-week mortality in chicks, thereby maintaining the production level. For that, constant monitoring of the bacterial populations is critical. Moreover, amidst the increased antibiotic resistance pattern, more focus on alternative treatment strategies like vaccines, probiotics, and bacteriophages is necessary. Future research focusing on strategies to mitigate APEC in broiler breeders would be one of the finest solutions for sustainable broiler production.

The Isolation and Characterization of Bacteriophages Infecting Avian Pathogenic Escherichia coli O1, O2 and O78 Strains.

Avian pathogenic Escherichia coli (APEC), such as O1, O2 and O78, are important serogroups relating to chicken health, being responsible for colibacillosis. In this study, we isolated and characterized bacteriophages (phages) from hen feces and human sewage in Alberta with the potential for controlling colibacillosis in laying hens. The lytic profile, host range, pH tolerance and morphology of seven APEC-infecting phages (ASO1A, ASO1B, ASO2A, ASO78A, ASO2B, AVIO78A and ASO78B) were assessed using a microplate phage virulence assay and transmission electron microscopy (TEM). The potential safety of phages at the genome level was predicted using AMRFinderPlus and the Virulence Factor Database. Finally, phage genera and genetic relatedness with other known phages from the NCBI GenBank database were inferred using the virus intergenomic distance calculator and single gene-based phylogenetic trees. The seven APEC-infecting phages preferentially lysed APEC strains in this study, with ECL21443 (O2) being the most susceptible to phages (n = 5). ASO78A had the broadest host range, lysing all tested strains (n = 5) except ECL20885 (O1). Phages were viable at a pH of 2.5 or 3.5-9.0 after 4 h of incubation. Based on TEM, phages were classed as myovirus, siphovirus and podovirus. No genes associated with virulence, antimicrobial resistance or lysogeny were detected in phage genomes. Comparative genomic analysis placed six of the seven phages in five genera: Felixounavirus (ASO1A and ASO1B), Phapecoctavirus (ASO2A), Tequatrovirus (ASO78A), Kayfunavirus (ASO2B) and Sashavirus (AVIO78A). Based on the nucleotide intergenomic similarity (<70%), phage ASO78B was not assigned a genus in the siphovirus and could represent a new genus in class Caudoviricetes. The tail fiber protein phylogeny revealed variations within APEC-infecting phages and closely related phages. Diverse APEC-infecting phages harbored in the environment demonstrate the potential to control colibacillosis in poultry.

Evaluation of the efficacy of two postweaning colibacillosis vaccines in a field herd with PRRS circulation during postweaning stage.

Postweaning diarrhea (PWD) and PRRS are two major concerns in swine production, which association has not been consistently explored. In the current scenario of restrictions in the use of antibiotics and ZnO, vaccination is more relevant to control PWD, but PRRS virus circulation may compromise the immune protection conferred by postweaning colibacillosis vaccines. We evaluated the efficacy of two postweaning colibacillosis vaccines (parenteral and oral) in a commercial herd affected by an outbreak of PWD and with PRRS circulation in postweaning. Five groups were studied during the postweaning period: one control (Group 1) and four vaccinated: two with each postweaning colibacillosis vaccine administered alone (Groups 2 and 3) or with sow vaccination against PRRS (Groups 4 and 5). We evaluated the effects on piglet weight, average daily weight gain and in the percentage of piglets with diarrhea, its duration, lethality and mortality. PRRS viremia and anti-PRRS antibodies were evaluated by qPCR and ELISA. Regarding control group, colibacillosis vaccination generally improved most of the measured parameters; but significant improvements were only observed in Groups 4 and 5 (p < 0.05). Moreover, cases of diarrhea occurred at different ages: in Groups 2 and 3 the peak of cases occurred just after ZnO was removed from the feed compared to Group 1, while in Groups 4 and 5 no peak was observed. This suggests that postweaning colibacillosis vaccination may be compromised by the PRRS circulation. In PRRS endemic herds an effective protection against PWD through vaccination may require PRRS vaccination to obtain a better performance.

In ovo administration of a phage cocktail partially prevents colibacillosis in chicks.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the main bacterial disease in poultry leading to significant economic losses worldwide. Antibiotic treatments favor the emergence of multidrug-resistant bacteria, and preventive measures are insufficient to control the disease. There is increasing interest in using the potential of bacteriophages, not only for phage therapy but also for prevention and biocontrol. This study aimed to evaluate the efficacy of a phage cocktail administered in ovo to prevent avian colibacillosis in chicks. When 4 different phages (REC, ESCO3, ESCO47, and ESCO58), stable under avian physiological conditions, were combined and inoculated at 17 embryogenic days (ED), they were transmitted to the newly hatched chicks. In a second trial, the 4-phage cocktail was inoculated into the allantoic fluid at ED16 and after hatch 1-day-old chicks were challenged with the O2 APEC strain BEN4358 inoculated subcutaneously. Two phages (REC and ESCO3) were still detected in the ceca of surviving chicks at the end of the experiment (7-days postinfection). Chicks that received the phages in ovo did not develop colibacillosis lesions and showed a significant decrease in intestinal BEN4358 load (8.00 × 107 CFU/g) compared to the challenged chicks (4.52 × 108 CFU/g). The majority of the reisolated bacteria from the ceca of surviving chicks had developed full resistance to ESCO3 phage, and only 3 were resistant to REC phage. The partially or complete resistance of REC phage induced a considerable cost to bacterial virulence. Here, we showed that phages inoculated in ovo can partially prevent colibacillosis in 1-wk-old chicks. The reduction in the APEC load in the gut and the decreased virulence of some resistant isolates could also contribute to control the disease.

Genetic Diversity and Antimicrobial Resistance of Extraintestinal E. coli Populations Pre- and Post-Antimicrobial Therapy on Broilers Affected by Colisepticemia.

The aim of the present study was to investigate the genetic diversity and antimicrobial resistance (AMR) of E. coli during enrofloxacin therapy in broilers affected by colisepticemia. Three unrelated farms with ongoing colibacillosis outbreaks were sampled at day 1 before treatment and at days 5, 10 and 24 post-treatment. A total of 179 E. coli isolates were collected from extraintestinal organs and submitted to serotyping, PFGE and the minimum inhibitory concentration (MIC) against enrofloxacin. PFGE clusters shifted from 3-6 at D1 to 10-16 at D5, D10 and D24, suggesting an increased population diversity after the treatment. The majority of strains belonged to NT or O78 and to ST117 or ST23. PFGE results were confirmed with SNP calling: no persistent isolates were identified. An increase in resistance to fluoroquinolones in E. coli isolates was observed along the treatment. Resistome analyses revealed qnrB19 and qnrS1 genes along with mutations in the gyrA, parC and parE genes. Interestingly, despite a fluoroquinolone selective pressure, qnr-carrying plasmids did not persist. On the contrary, two conjugative AMR plasmid clusters (AB233 and AA474) harboring AMR genes other than qnr were persistent since they were identified in both D1 and D10 genomes in two farms. Further studies should be performed in order to confirm plasmid persistence not associated (in vivo) to antimicrobial selective pressure.

Antimicrobial Efficacy of Cinnamon Essential Oil against Avian Pathogenic Escherichia coli from Poultry.

Colibacillosis, caused by E. coli, is responsible for economic losses in the poultry industry due to mortality, decreased production, and the cost of antibiotic treatments. Prevention of colibacillosis is based on improved biosecurity measures and the use of the vaccine performed with O78 E. coli strains, which is responsible for most cases of colibacillosis. Recently, there has been increased interest in other infection control methods, such as the use of natural compounds. The aim of this study was to evaluate the antimicrobial efficacy of cinnamon essential oil (CEO) against E. coli strains isolated from poultry. The MIC50 and MIC90 of CEO were determined by testing 117 strains belonging to serogroups O78, O2, O128, O139, isolated from laying hens (91 strains), broilers (10 strains), and turkeys (16 strains). The bacterial strains were tested at cell densities of 108 and 106 CFU/mL. At the cell density of 108 CFU/mL, MIC50 and MIC90 were 0.4 and 0.5 µL/mL for most of the tested strains, while they corresponded to 0.5 µL/mL for all strains isolated from broilers and for strains belonging to serogroup O139. At the cell density of 106 CFU/mL, MIC50 and MIC90 were 0.3 and 0.4 µL/mL, regardless of bird species of origin and for strains belonging to serogroups O78 and O2. In addition, a concentration of 0.04 µL/mL of CEO corresponded both to MIC50 and MIC90 for strains belonging to serogroups O139 and O128. Based on these results, cinnamon essential oil showed an effective antibacterial activity against E. coli strains from poultry and could find field application for the prevention of colibacillosis.

Bird immunobiological parameters in the dissemination of the biofilm-forming bacteria Escherichia coli.

Background and Aim: With the development of industrial maintenance technology, a group of pathogens called avian pathogenic Escherichia coli (APEC) became very common. The initiation, development, and outcome of the infectious process mediated by virulent APEC strains occur through a decrease in the colonization resistance of the intestine, an immunobiological marker of homeostasis stability in susceptible species. This study focused on the pathogenetic features of colibacillosis and the morphological features of E. coli. Materials and Methods: Clinical, immunological, bacteriological, and histological studies were conducted on 15-day-old white Leghorn birds (n = 20). The birds were divided into two groups: Control group (Group I; n = 10) with birds intranasally inoculated with 0.5 mL of 0.9% NaCl solution and experimental group (Group II; n = 10) with birds intranasally inoculated with 0.5 mL of an E. coli suspension at 1 billion/mL. Results: During the biofilm formation, clusters of microcolonies were formed as a gel-like intercellular matrix that accumulated due to cell coagulation. The intercellular matrix "glues" heteromorphic cells together and forms a structure of densely packed heteromorphic cells arranged in an orderly manner and growing in different directions. During the experimental reproduction of E. coli, excessive growth was observed in material isolated from poultry. Pathogenic E. coli strains implementing virulence factors adhered to the receptors of erythrocytes, alveolocytes, and enterocytes. Multicellular heterogeneous biofilms, united by an intercellular matrix, were located at the apical poles of the respiratory tract alveolocytes and enterocytes of the terminal ileum villi. Many bacteria exudate containing desquamated epithelial cells with an admixture of mucus, and polymorphonuclear leukocytes were detected in the lumen of the birds' abdominal organs. Invasive bacteria damaged the epithelial layer, violated the endothelial layer of blood vessels, and developed inflammatory hyperemia of the lamina propria of the respiratory and digestive systems' mucous membrane. A correlative dependence of changes developed by the type of delayed hypersensitivity reaction was established. Signs of accidental transformation of the thymus, atrophy of the bursa of Fabricius, disseminated thrombosis, and septic spleen developed. Moreover, toxic cardiomyocyte dystrophy, signs of congestive vascular hyperemia, massive disintegration of lymphocytes, macrophage reactions, perivascular edema resulting from the release of plasma, and shaped blood elements were detected. Conclusion: The development and outcome of the infectious process in escherichiosis primarily depend on the homeostasis stability of susceptible species and virulence factors of the pathogenic microorganisms. One of the selected strains, E. coli O78:K80 displayed the highest ability to form biofilms. Its strong adhesion ability to bird erythrocytes was demonstrated. Deepening the scientific knowledge of the interaction between eukaryotes and prokaryotes will contribute to a better understanding of the pathogenetic aspects of avian escherichiosis and eventually find promising anti-adhesive drugs that could reduce primary bacterial contamination in vivo and in vitro.

Antimicrobial Efficacy of Phyto-L, Thiosulfonate from Allium spp. Containing Supplement, against Escherichia Coli Strains from Rabbits.

Colibacillosis, caused by enteropathogenic Escherichia coli (EPEC), is one of the most common diseases in rabbit farms, resulting in economic losses due to mortality and decrease in production. Until recently, antimicrobials were used to both treat and prevent disease on livestock farms, leading to the possible risk of antimicrobial resistance (AMR) and the selection of multidrug-resistant (MDR) bacteria. Therefore, interest in alternative control methods, such as the use of natural substances, has increased in the scientific community. The aim of this study was to evaluate the antimicrobial efficacy of Phyto-L (Pro Tech s.r.l.), a product containing organosulfur compounds (OSCs) such as propyl propane thiosulfonate (PTSO) from Allium spp., against 108 strains of E. coli isolated from rabbits with colibacillosis from 19 farms. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Phyto-L were assessed. Bacterial suspensions with a charge of 108 CFU/mL, corresponding to those found in the rabbit gut under pathologic conditions, were tested with different concentrations from 20 to 0.15 µL/mL of Phyto-L. For each strain, the MIC and concentrations above the MIC were plated on Tryptic Soy agar (TSA) without Phyto-L to assess the MBCs. MIC and MBC values ranged from 1.25 to 5 µL/mL and 1.25 to 20 µL/mL, respectively, depending on the strain tested. The data showed an interesting antibacterial activity of Phyto-L against EPEC strains. Therefore, this product could be effective in preventing colibacillosis in field application, especially considering that 104-105 CFU/g of feces is the amount of E. coli usually found in the gut contents of rabbits under physiological condition.