High genetic diversity among extraintestinal Escherichia coli isolates in pullets and layers revealed by a longitudinal study

An infection with E. coli in layers is regarded as one of the major problems in global poultry industry that might cause reproductive disorders referred as salpingitis/peritonitis/salpingoperitonitis and peritonitis syndrome ultimately leading to severe economic losses on commercial farms [6]. In this regards, an epidemiological knowledge of the disease and disease causing agent is fundamental in order to develop effective control and prophylactic strategies. Here, we studied molecular epidemiology of E. coli isolates collected from pullets and layers in a longitudinal sampling study in Austria. Data obtained from genetic fingerprinting by PFGE were analyzed together with serotypes, geographical regions of isolation, and concurrent pathological lesions in each of the sampled birds.

In total, more than half of the E. coli isolates (n?=?91/144) could not be assigned to a single serotype using antibodies against O1:K1, O2:K1 and O78:K80. Furthermore, for those isolates that could be assigned to one of the named serotypes, no correlation was found between a specific serotype and the occurrence of lesions in birds. In previous studies, it was also shown that E. coli isolates collected from diseased birds display a high serological diversity [16, 21, 22], demonstrating as high as 62 different O serogroups [21]. Thus classifying E. coli strains into a definite serotype might sometimes be somewhat challenging. Hence, our finding is in agreement with a previous notion that serotyping alone might not be helpful as a tool for characterization of E. coli [16].

In this study, the PFGE subtyping of E. coli isolates (n?=?132) resulted in 96 XbaI profiles. Exclusively in two events, the same PFGE profile was seen in isolates from different sampling dates in mutually related farms/flocks, indicating potential E. coli persistence. The PFGE-type S7 (n?=?3) included isolates from pullets (n?=?2, rearing farm R_V) without pathological lesions and from one layer in the corresponding flock L_1/V suffering from egg peritonitis and fibrinous oophoritis at the peak of production. In the second case, PFGE type S32 contained two isolates from the same layer flock (L_2/VI) at the peak and end of production. One bird sampled at the peak of production showed inflammation of the ovary whereas egg peritonitis was diagnosed in the other birds necropsied at the end of production. These results indicate that some E. coli genotypes may retain in certain flocks at different stages of rearing but the associated pathological outcomes in birds can vary.

The genomic profile of extraintestinal E. coli with PFGE further revealed that strains collected from birds with pathological lesions can have 100 % genetic identity with strains that were collected from healthy birds. For instance, in PFGE type S10 (n?=?3) in flock L_2/V, two birds did not have any lesions while one had oophoritis and salpingitis. Likewise in PFGE type B7 (n?=?2) in L_2/III, one bird showed no lesions while in contrast, the other had egg peritonitis. Also, remaining isolates could not be grouped into distinct clonal clusters based on presence or absence of pathological lesions in sampled birds. This finding is in agreement with a previous study in broilers where authors have reported a high heterogenecity of E. coli isolates in broilers [13, 23]. It can be hypothesized that pathogenicity of extraintestinal E. coli in chickens is highly dependent on concurrent environmental and host susceptibility factors. Providing a suitable opportunity in certain circumstances, E. coli residing in clinically healthy chickens might turn up into pathogenic. The hypothesis is further supported by an earlier finding in broiler that many collibacillosis associated isolates might not be clearly distinguished solely on the basis of presence of virulence associated genes as compared to intestinal commensal E. coli [13].

In the present study, we found no evidence for clonality of E. coli with respect to geographical locations of farms. Previously, Ewers et al. (2004) found only a limited number of E. coli clones to be distributed in poultry production in Germany [16]. In another study, it was reported that chickens with peritonitis in a single flock were likely to be infected by the same E. coli strain [24]. Different to this, we did not find clonality of E. coli isolates in birds from the same flock showing gross pathological lesions in the reproductive tract thus maintaining a high heterogenicity of PFGE types. Interestingly, we further noticed that a single bird can harbour two different PFGE types of E. coli in the same or different organs. Thus, the study demonstrated that a layer can be infected simultaneously by different E. coli genotypes. A similar finding was previously reported in broilers [18]. However, in another study in layers, one PFGE type was found to be present in bone marrow of an individual bird [17]. It might be that in some organs E. coli isolates possess less or no genetic diversity due to an adaptation process, which should however be further elucidated. In the present study, we also tested antibiotic susceptibility of 16 isolates that were collected from eight birds. All the isolates were sensitive to ceftiofur, colistin, gentamicin and spectinomycin but the resistant rate to tylosin was found 100 %. Mixed results were obtained for other antibiotics tested. MDR was seen in 3/16 isolates showing resistance to as high as five different antibiotics used. Although the number of isolates included for antimicrobial susceptibility test in the actual study is not very high, it already provides an indication for the problem of antibiotic resistance in E. coli towards commonly used antimicrobials. In a recent report from China, E. coli isolates collected from chickens were sensitive to relatively newer antibiotics such as cephalosporin but MDR rate was as high as 80.25 % [11]. The results from the present study further indicate that isolates collected from the same bird may not necessarily have identical antibiotic sensitivity profiles. Thus it can be suggested that testing of the antibiotic sensitivity profile from just one isolate per bird might not be enough to decide the most appropriate treatment.