Escherichia coli in broiler chickens in Egypt, its virulence traits and vaccination as an intervention strategy

Avian pathogenic Escherichia coli (APEC) is one of the extra intestinal pathogenic E. coli (ExPEC). Previous studies showed that O 1 , O 2 and O 78 serotypes are mostly associated with Colibacillosis outbreaks, but recently there are emergent new pathogenic serotypes that have spread worldwide. Wide antigenic diversity exists among APEC strains in Egypt; however, the involvement of a particular O serotype in the infection process appears to vary with the geographical region. Different virulence genes have been identified in APEC. Recently; the presence of these virulence genes is being employed as an indication of pathogenicity, rather than the tedious E. coli serotyping methods. In Egypt; several virulence genes were studied, and were found to be different based on the geographical area. However; all studies were limited to a small number of screened virulence genes, in addition to the inconsistency of these screened genes. To control APEC, antibiotics have been used for decades; however the emergence of multi-drug resistant E. coli , and the difficulty of discovering new antimicrobial therapies made vaccine the best choice to control E. coli infections in poultry farms. In this review, the various aspects of APEC infection in poultry with special focus on the epidemiology of APEC in Egypt in relation to virulence traits were discussed. In addition, the most recent vaccination trials against the APEC diseases in poultry were discussed. We concluded that the virulence gene patterns of APEC can be considered as molecular markers of pathogenicity. Although of their current limitations, some vaccine trials showed promising results as good alternative to control colibacillosis in poultry.


Introduction
E. coli is a Gram-negative, rod-shaped, facultative anaerobic bacterium of the Enterobacteriaceae family.
Colibacillosis in poultry includes systemic and localized infections. The localized infections were; omphalitis, swollen head syndrome, cellulitis, and diarrhea. Whereas systemic infection including; respiratory colisepticemia, enteric colisepticemia, and neonatal colisepticemia. Ewers et al., (2003) reported that E. coli pathogenicity was generally enhanced or initiated by several influencing factors such as; environmental factors, viral infections, mycoplasma infections, and immune-suppression.
Susceptibility to APEC infection increased after exposure to many intrinsic and extrinsic factors. Extrinsic factors involve environment; exposure to other infections, virulence, duration and levels of exposure, whereas intrinsic factors involve age; route of exposure, passive and active immune status, in addition to strain and breed of chickens. Generally, young birds were more susceptible to sever infections than adult (Rodriguez-Siek et al., 2005). Horizontal infections occurred with E. coli by contact with other birds, in addition to feacal and oral routes. Nolan et al., (2013) added that false vertical transmission of E. coli was reported from breeders through egg shell contamination during hatching, or in ovo due to salpingitis.
Diagnosis of colibacillosis in chicken broilers was based on the clinical signs and the typical lesions. Moreover; molecular techniques could be used for phenotypic characterization of the bacterial isolates, by using specific Polymerase Chain Reactions (PCRs).
Pathotypes of E. coli can be further identified depending on the presence of virulence genes (VGs). Currently; we review the various aspects of APEC infections in poultry, with special focus on the epidemiology of APEC in Egypt in relation to virulence traits. In addition, the most recent vaccination trials against the APEC diseases in poultry were discussed.

Avian pathogenic E. coli infection in poultry
Early studies on avian E. coli strains showed that O 1 , O 2 O 15 , O 35 and O 78 serotypes, were mostly associated with colibacillosis outbreaks (Dho-Moulin and Fairbrother, 1999). Later study of Nolan et al., (2013) Younis et al., (2017);El-Sawah et al., (2018) studies showed that wide antigenic diversity existed among avian pathogenic E. coli strains in Egypt, and worldwide. Thus, the involvement of a particular O serotype in the infection process appeared to vary according to the geographical region (Table 1).

Serum resistance
Serum resistance VGs allow the bacteria to survive exterior to the gastrointestinal tract, and overcome defense mechanisms of the host involving complement and antimicrobial peptides. Mellata et al., (2003) reported that the capsular K 1 and somatic O 78 polysaccharide increased serum resistance of the APEC, which can lead to bacteremia.
A study of Nilsson et al., (2014) showed a strong correlation between the APEC pathogenicity and four serum resistance VGs such as; increased serum survival (iss), structural genes of Colicin V operon (cvaC), surface exclusion protein (traT), and outer membrane protein A (ompA). The iss was significantly associated with APEC than AFEC. However; individual existence of the iss gene\ or whether this iss Novel Research in Microbiology Journal, 2019 gene was a marker gene for the presence of the plasmids correlating with APEC pathogenicity was not confirmed.

Adhesions
Bacterial adhesion is based on recognition between bacterial surface components and specific receptors in host tissues. ExPEC strains encode many adhesions that promote the attachment of the bacteria to cell receptors, and were very important for development of septicemia (Monroy et al., 2005).
Type 1 fimbriae (F 1 fimbriae) have been involved with the initial stages of upper respiratory colonization, whereas the P fimbriae were involved in colonization of the internal organs. The F 1 fimbriae were encoded by a total of nine fim genes which include a major protein named as FimA, and minor proteins named as FimF, FimG and FimH adhesions. Nevertheless, earlier study of Arne et al., (2000) showed that the APEC fimH mutant strain failed to adhere to the chicken trachea epithelial cells in vitro. The P fimbriae are hem-agglutinating fimbriae with mannose resistant properties; were found in E. coli strains producing human urinary tract infections as well as some APEC. Moreover; they were linked to the colonization of internal organs, which led to septicemia and lethality in one-day-old chickens. P fimbriae were encoded by pyelonephritis associated pili gene clusters (pap). This pap gene cluster involved eleven genes (papI, papB, papA, papH, papC, papD, papJ, papK, papE, papF, papG), for the biogenesis and synthesis of the P fimbriae (Dozois et al., 2000).
Curli fimbriae type are thin and curly appendices found on the cell surface of Salmonella enterica and E. coli; and were responsible for the bacterial linkage to proteins of the extracellular matrix, causing survival of such bacteria in the external environment. According to La Ragione and Woodward, (2002), the genes accountable for curli fimbriae expression were encoded by two types of operons: csgBAC and csgDEFG. CsgA sequence was recognized only in all APEC, recovered from chickens suffering from septicemia (Amabile de Campos et al., 2005). Additional adhesions recognized between APEC strains and suggested to be involved in the pathogenesis of these strains include; type 1-like fimbriae, AC/1 fimbriae, Afa, Sfa, F 17 , and Eae fimbriae related sequences (McPeake et al., 2005).

Iron acquisition systems
Iron is essential for the persistence of bacteria due to its involvement to many cellular activities Novel Research in Microbiology Journal, 2019 including; nucleotide biosynthesis, peroxide reduction, and electron transport. Iron acquisition systems among APEC strains may be encoded by plasmid genes, or by chromosomal pathogenicity islands (Johnson et al., 2006). The common iron sequestering mechanism in iron deficient host environments is the siderophores production. In APEC, the aerobactin operon area encodes five polypeptides. Four genes (iucABCD) encode for polypeptides that contribute in aerobactin synthesis; in addition to one gene (iutA) that encodes for an outer membrane protein, which serves as a receptor (Carbonetti and Williams, 1984). Zhu et al., (2005) revealed that Salmochelins siderophores system; which were the first discovered siderophores identified in Salmonella enterica, comprised five genes (iroB, iroC, iroD, iroE, and iroN), that have been described among APEC. The iroN gene encoding for an outer membrane siderophore, was considered as the chief receptor for transport of ferric salmochelin (Hantke et al., 2003). In spite of being located on plasmids, the salmochelin and aerobactin encoding operons were controlled by the chromosomally located fur gene product. This gene inhibits siderophores production when sufficient quantities of free iron were existing in the environment (Balbontín et al., 2016).
A study of Paixão et al., (2016) reported the existence of an association between the siderophore yersiniabactin firstly detected in Yersinia enterocolitica (encoded by irp-2 (iron-repressible) and fyuA (ferric yersinia bactin uptake genes), and pathogenicity of APEC.

Temperature-sensitive hemagglutinin and colicins
The temperature-sensitive hemagglutinin (tsh) gene is an auto transporter protein with double functions of proteolytic and adhesive activities. This protein stays in the outer membrane and helps the adhesion process during the early stages of the infection. Generally, this gene was identified on ColV plasmids at a greater frequency among APEC (Nakazato et al., 2009). The tsh gene was an important virulence markers of APEC having a strong association with internal organs colonization; septicemia and lethality in one-day-old chickens (Ngeleka et al., 2002), which made it a usefl target for pathotying of APEC.
Colicins are minor protein molecules secreted by E. coli; which were classified as bacteriocins because of their antibacterial activity toward some species of bacteria (Cascales et al., 2007). A study of Dias da Silveira et al., (2002) presented E1, E2, E3, I, K, B, Ia, Ib and V as the most predominant colicins in APEC isolates. Most of APEC strains have colicin V plasmids which harbor other pathogenicity associated genes.
The existence of ehxA, sat and cnf1 genes have also been described in APEC strains (da Silva et al., 2017). However, their function in pathogenesis was not fully clarified. Additionally; some of the toxins genes (hly, cdt and cvaC) have been associated with large transmissible plasmids, indicating that these VGs might be easily transmitted to other strains (Mellata et al., 2012). Recent study of Murase et al., (2016) suggested that hlyF which was one of the genes of the ColV plasmid, as a molecular indicator for APEC.
Novel Research in Microbiology Journal, 2019 Moreover, this gene was directly included in the outer membrane vesicles production.
Shiga toxin gene had been detected in avian E. coli by PCR, but the proof of its expression was little. Lately; a strong mediator for apoptosis (caspase 3/7induced) and cytotoxic action was described, following a 6-h infection assay using macrophage cell line by an APEC strain (Bastiani et al., 2005). Other toxins described in APEC strains involved the heatlabile enterotoxin and the heat-stable enterotoxin 1 (AstA), homologue of entero-aggregative E. coli (Janben et al., 2001).
In Egypt, several virulence genes screened; however, the main limitation of all studies was the limited number of virulence genes screened in each study, and the inconsistency of the screened genes (Table 2).

Current status of vaccine development against APEC
Expanded antibiotics resistant E. coli are posing a zoonotic risk to humans. Meanwhile; the careless use of antimicrobials in the developing countries, and the difficulty of discovering new antimicrobial therapies for resistant E. coli, led to the suggestion of using the vaccines as the best choice to control E. coli infections in poultry farms. Multiple trials have been conducted for evaluating the efficacy of using vaccination against E. coli infecting poultry. However, several difficulties hindered such efforts including; the capability of the vaccine to induce cross protection against various APEC sero-groups, vaccine mass delivery method, and timing of vaccination (Ghunaim et al., 2014).
In Table 3, some trials for development of E. coli vaccines in poultry in the last five years are summarized. Generally, studies revealed that the inactivated vaccines provided protection against homologous challenges only (Roland et al., 2004).
Meanwhile, researches on live attenuated E. coli vaccines resulted in the production of two commercial vaccines. Both vaccines are currently used in Egypt; however, their field efficacy against homologous and heterologous E. coli need to be further evaluated (Galal et al., 2018). Although subunit vaccines demonstrated better immune response and better protection against homologous and heterologous challenges; however, large scale experiments were not conducted.  (Roland et al., 1999;2004

Conclusion
The epidemiology of E. coli serotypes in broiler chickens vary according to the geographical region in Egypt, and worldwide. The presence of individual virulence gene was not inductive to E. coli pathogenicity, rather than the existence of certain traits of these genes together. However, the inconsistency and incomplete screening of various virulence traits of the isolated E. coli in Egypt made it difficult to conclude specific virulence gene traits of the APEC. Finally, vaccines are promising strategy to control E. coli infections in the presence of multi-drug resistant strains; however, the availability of vaccines that provide cross protection against different APEC strains needs further investigation.

Conflict of interest
The authors declare that there is no conflict of interests.