Description and validation of a new set of PCR markers predictive of avian pathogenic Escherichia coli virulence.

Avian colibacillosis is the main bacterial infectious disease in poultry and is caused by avian pathogenic Escherichia coli (APEC). However, E. coli strains are very diverse, and not all are pathogenic for poultry. A straightforward scheme for identifying APEC is crucial to better control avian colibacillosis. In this study, we combined high-throughput PCR and a machine learning procedure to identify relevant genetic markers associated with APEC. Markers related to phylogroup, serotype and 66 virulence factors were tested on a large number of E. coli strains isolated from environmental, faecal or colibacillosis lesion samples in 80 broiler flocks. Nine classification methods and a machine learning procedure were used to differentiate 170 strains presumed non-virulent (obtained from farm environments) from 203 strains presumed virulent (obtained from colibacillosis cases on chicken farms) and to develop a prediction model to evaluate the pathogenicity of isolates. The model was then validated on 14 isolates using a chick embryo lethality assay. The selected and validated model based on the bootstrap aggregating tree method relied on a scheme of 13 positive or negative markers associated with phylogroups (arpA), H4 antigen and virulence markers (aec4, ETT2.2, frzorf4,fyuA, iha, ireA, iroN, iutA1, papA, tsh, and vat). It had a specificity of 84 % and a sensitivity of 85 %, and was implemented as an online tool. Our scheme offers an easy evaluation of the virulence of avian E. coli isolates on the basis of the presence/absence of these 13 genetic markers, allowing for better control of avian colibacillosis.

Efficacy of passive immunization in broiler chicks via an inactivated Escherichia coli autogenous vaccine administered to broiler breeder hens.

Avian pathogenic Escherichia coli (APEC) cause extra-intestinal infections called colibacillosis, which is the dominant bacterial disease in broilers. To date, given the diversity of APEC strains and the need for an acceptable level of protection in day-old chicks, no satisfactory commercial vaccine is available. As part of a French nationwide project, we selected three representative strains among several hundred APEC that cause colibacillosis disease. We first performed experiments to develop colibacillosis in vivo models, using an inoculum of 3 × 107 CFU of each E. coli strain per chick. Two APEC strains (19-381 and 19-383-M1) were found to be highly virulent for day-old chicks, whereas the third strain (19-385-M1) induced no mortality nor morbidity.We then produced an autogenous vaccine using the (Llyod, 1982; MaCQueen, 1967) 19-381 and 19-383-M1 APEC strains and a passive immunization trial was undertaken. Specific-pathogen-free Leghorn hens were vaccinated twice 2 weeks apart, the control group receiving a saline solution. The vaccinated and control hens exhibited no clinical signs, and egg production and fertility of both groups were similar. Fertile eggs were collected for 2 weeks after the second vaccination and chicks were obtained. After challenge with each APEC (19-381 and 19-383-M1), chicks appeared to be partially protected from infection with the 19-383-M1 strain, with 40% mortality compared with 80% for the non-vaccinated chicks. No protection was found when the chicks were challenged with the 19-381 strain. Now, further work is needed to consider some aspects: severity of the pathogen challenge model, persistence of the protection, number of APEC strains in the autogenous vaccine, choice of adjuvants, and heterologous protection by the vaccine made from strain 19-383-M1.RESEARCH HIGHLIGHTS Three APEC strains were characterized and selected to develop in vivo models of colibacillosis.A bivalent autogenous vaccine was produced and a passive immunization trial was carried out.Protection of chicks was demonstrated when challenged with the 19-383-M1 APEC strain (homologous challenge).Further work is needed in particular to evaluate the protection against heterologous challenge.

Storage Temperature or Thermal Treatments During Long Egg Storage Duration Influences Hatching Performance and Chick Quality.

This study was designed to improve the hatching performance, chick robustness and poultry health in the event of long-term egg storage and suboptimal age of the reproductive flock. A total of 9,600 eggs from one young breeder flock (28 weeks of age, batch B) and 9,600 eggs from an older breeder flock (59 weeks of age, batch E) were used (ROSS 308). Each batch was separated into three sub-groups and stored for 14 days. The first sub-group of eggs (Cool, group C) was stored at 11.6°C. The second sub-group of eggs (Warm, group W) was stored at 18.3°C with two pre-incubation on days 6 and 10 of the storage period. The final sub-group of eggs (Control, group Ct) was stored at 18.3°C throughout the storage period. Eggs were similarly incubated and hatched birds were raised on the same experimental farm. In both batches, embryonic development was significantly more advanced in W eggs than in C and Ct eggs ( p < 0.01). In both batches, C and W treatments decreased early embryonic mortality by more than 10% compared with Ct, decreased the proportion of late-hatched chicks and improved the percentage of first grade chicks: in batch E, 42% of Ct eggs were first grade chicks vs. 57% in group W and 59% in group C. Benefits were even higher in batch B, where only 60% of Ct eggs gave first grade chicks vs. 83% in others groups. The hatching rate was thus higher in groups C and W regardless of flock age: for batch B eggs, 85% hatched in W and 84% in C vs. 62% in Ct, while for batch E eggs, 59% hatched in W and 61% in C vs. 45% in Ct. Day-old Ct chicks from batch E were heavier than W and C ones, and heavier than W chicks from batch B ( p < 0.05). Long-term parameters on farm were not significantly different between groups. Thermal treatments during the storage of eggs from both young and old breeder flocks counterbalance the negative effects of prolonged egg storage on hatching rate, without altering chicken performance during rearing.

Diversity of Escherichia coli strains isolated from day-old broiler chicks, their environment and colibacillosis lesions in 80 flocks in France.

Avian colibacillosis is the most common bacterial disease affecting broilers. To better evaluate the diversity and the origin of the causative Escherichia coli strains infecting birds, we conducted a study on 80 broiler flocks. Just before the arrival of chicks on the farm, samples were collected in the farm environment (walls, feeders, air inlets, etc.) and, upon delivery, day-old chicks (DOCs) and the transport boxes were also sampled. Isolates were obtained from these samples, and from organs of chickens exhibiting typical colibacillosis symptoms. The isolates were characterized using high-throughput qPCR to detect a range of genetic markers (phylogroups, main serogroups virulence markers, etc.). A total of 967 isolates were studied, including 203 from 28 colibacillosis episodes, 484 from DOCs, 162 from transport boxes and 118 from the farm environment. These isolates yielded 416 different genetic profiles, of which 267 were detected in single isolates, and the others were observed in up to 44 isolates from nine farms. The distributions of isolates across phylogroups and the main serogroups varied with the origin of isolation. The isolates obtained from colibacillosis cases either shared a single genetic profile or were different. In a few cases, we observed the same profile for isolates obtained from DOCs and colibacillosis lesions in the same flock or different flocks. However, some flocks receiving DOCs contaminated with isolates bearing the genetic profile of colibacillosis cases identified in other flocks remained healthy. This study highlights the huge diversity among avian E. coli isolated from diseased and non diseased birds.

Dust exposure and health of workers in duck hatcheries.

OBJECTIVES: The objectives of this cross-sectional study were to investigate dust exposure and respiratory health of workers in duck hatcheries in western France. MATERIAL AND METHODS: Ninety volunteer workers, who work in sorting rooms and/or incubation rooms, participated in exposure assessments and medical examinations. Medical examinations were performed by occupational health practitioners.They filled-in a questionnaire with the workers, followed by a lung function test on each worker. General characteristics and prevalence of chronic respiratory symptoms were described in each type of working rooms. Associations between symptoms and exposure (working room or dust level) were studied in GEE multivariate models. RESULTS: Overall prevalence of chronic respiratory symptoms (cough, phlegm) and chronic bronchitis were similar or lower than in the reference population. However, prevalence of these symptoms was higher for those working in sorting rooms, that were associated with an increased risk of respiratory symptoms and decreased lung function. Respirable dust was also significantly associated with an increased risk of respiratory symptoms. The prevalence of asthma and rhinitis were well above those in the reference population, but did not vary among working rooms. Descriptive data suggested an occupational origin for some cases. CONCLUSIONS: Hatchery workers were at increased risk of compromised respiratory health due to dust exposure, particularly those who work in sorting rooms. Asthma and rhinitis were in excess in this population of workers. Thorough clinical examination of these workers should be performed and all exposures assessed.