Virulence characterization of avian enterococcus faecalis field isolates and genetic approach to select more resistant laying hens

Resumen

In the last twenty years, major progress in the understanding of systemic amyloid-A amyloidosis, also known as amyloid arthropathy (AA) in chickens, has been gained. However, several topics related to this disease remain unclear, such as the higher susceptibility of brown layers compared to white layers, the biochemical mechanism of the pathogenesis and the transmission route as well as a possible effective treatment to prevent or cure the disease. AA is the only type of amyloidosis that has been associated with chronic infections induced by Enterococcus (E.) faecalis strains and, therefore, the treatment of this disease has focused to date on the prevention and/or treatment of this infection. Given the intrinsic and acquired resistance of E. faecalis to several antimicrobials and the new regulations to reduce the antibiotic use in animal production, its treatment becomes more complicated. Additionally, little is known about the risk from field avian E. faecalis strains and it is currently difficult to estimate its virulence degree. Consequently, control measures against E. faecalis infection such as biosecurity, hygiene, responsible use of antibiotics, development of effective vaccines or the breeding and genetic selection are essential. Therefore, in the present dissertation different avian E. faecalis field strains were characterized and the possibility to select more resistant laying hens against E. faecalis infection through the Embryo Lethality Assay (ELA) was investigated. The ELA is proposed as an alternative model to replace the adult avian challenge assays. To date, adult avian challenge assay is the most used animal model in order to determine the degree of virulence of isolated fungi and bacteria, as well as in the selection for disease resistance. However, this model is currently limited by the exposure to pathogens on a large scale, its technical complexity, the cost associated and for ethical reasons. Therefore, the aim of the Experiment 1 was to develop a generic methodology of the ELA as a diagnostic model to establish the pathogenicity of E. faecalis strains in embryos of White Leghorn layers. Of particular interest was to determine the virulence of the avian E. faecalis strain K923/96 and assess its median lethality dose (LD50) in order to use it as reference strain in further analyses. The ELA was repeated four times. 0.2 ml of different infectious doses of the cited strain (from 2.5 to 2500 colony-forming units (CFU) per ml) were inoculated into the allantoic cavity (AC) of 10-day-old embryos. The embryo mortality rate (EMR) was determined by daily candling of the eggs over a period of seven days and based on this information the LD50 was calculated. The infectious dose showed a significant effect on the EMR. The EMR with the doses of 2.5, 5, 25, 250 and 2500 CFU/ml was 43%, 45%, 63%, 90% and 93%, respectively. The estimated dose at LD50 was 6.6 CFU/ml. The highest EMR was recorded after three and four days post-inoculation (p.i.) in all doses. In summary, the methodology proposed in this experiment can be used as reference in further researches on E. faecalis virulence taking into account that the EMR produced in the ELA depends on the dose; the higher the infectious dose, the greater the EMR and the lower the embryo survival time. In order to characterize the virulence of different avian E. faecalis field strains and thus corroborate the ELA capacity for distinguishing E. faecalis strains, 68 avian E. faecalis strains through the ELA were characterized in the Experiment 2. Of particular interest was to characterize the genetic diversity of these strains by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) under the hypothesis that strains with similar DNA banding patterns might be similar also in its infective ability. The ELA was performed ten times with subsets of 7-8 E. faecalis strains on a sample of 9987 eggs, including two control groups (positive and negative). An estimated 3-24 CFU were inoculated into the AC of 10-day-old embryos of White Leghorn layer hens. The EMR was determined by means of candling the eggs over a period of seven days. The ELA was able to distinguish the virulence of the E. faecalis strains. Twenty-six strains were considered as avirulent strains with an EMR of below 40%. Five strains were highly virulent with an EMR above 80%, and the remaining 37 strains were classified as strains of moderate virulence, causing an EMR between 40% and 80%. The highest EMR occurred three and four days p.i. From the fourth day p.i., almost no embryonic mortality was observed. Therefore, the ELA could be optimized by reducing experiment duration to four days p.i. ERIC-PCR did not cluster the strains according to its virulence, although ERIC banding patterns revealed a considerable genetic diversity. In conclusion, the ELA can be considered a reliable and useful tool to predict the virulence of avian E. faecalis strains. The results obtained in this experiment can be used as a basis for future researches on the E. faecalis virulence, and high pathogenic isolates tested might be good candidates for the production of vaccines. The aim of the Experiment 3 was to investigate the possibility to select more resistant laying hens against E. faecalis infection through the ELA, which is proposed as an alternative model to replace the adult avian challenge assays. A preliminary study was first carried out under the hypothesis that chicken embryos of brown layers would be more susceptible compared with embryos of white layers when they are infected with the same E. faecalis strain and infectious dose since white layers are less susceptible to AA than brown layers. To this end, the ELA was performed six times in the preliminary study. 0.2 ml of two infectious doses of the avian E. faecalis strain K923/96 (2.5 and 5 CFU/per ml) were inoculated into the AC of 10-day-old embryos of brown layers. The EMR was determined by daily candling of the eggs over a period of four days. The infectious dose showed a significant effect on the EMR. The average EMR estimated for the doses of 2.5 and 5 CFU/ml was 69% and 83%, respectively. They differed significantly from each other. On the basis of these results, 7563 embryos of 10-day-old from 500 families of brown layers (from one to eight embryos per family) were inoculated into the AC with only one infectious dose of 2.5 CFU/ml of the avian E. faecalis strain K923/96 in three more ELAs. The EMR was determined by daily candling of the eggs over a period of four days. Genetic parameters of embryonic survivability to E. faecalis infection were estimated to judge the possibility to apply the value of genetic selection. Furthermore, the genetic correlation between embryonic survivability and some traits of economic interest such as laying performance, egg weight (EW), body weight (BW), breaking strength (BS) and dynamic stiffness (Kdyn) was calculated. The average EMR estimated of these three ELAs was 50% and the highest EMR occurred three and four days p.i. as expected. The estimated heritability for embryonic survivability to the infection by E. faecalis was h2 = 0.17 – 0.20 indicating that the selection of more resistant laying hens to E. faecalis infection is feasible. However, a high genetic negative correlation was estimated between embryonic survivability and laying performance at the peak of lay (rg= -0.69) and at the end of the production (rg= -0.55), as well as with BS (rg = -0.48 to -0.30), which is undesirable from a breeding point of view. A positive correlation was found between EW and the embryonic survivability (rg= +0.16) and no genetic correlation with BW and Kdyn was found. Therefore, although it was demonstrated that it is possible to breed for E. faecalis resistance, the improvement of other important traits will be penalized. On the basis of the results obtained in the present dissertation it can be concluded that the ELA is a reliable and useful tool to characterize the virulence of avian E. faecalis strains as well as to select more resistant laying hens against E. faecalis infection, which can be use as alternative model to replace the adult avian challenge assays. Virulence characterization of avian E. faecalis field isolates allows us to know the risk that the strains represent and provide valuable information, which can be important for the selection of isolates for the production of vaccines. Regarding to the possibility to select more resistant laying hens against E. faecalis infection, the estimated heritability for embryonic survivability to the infection of E. faecalis indicates that it is feasible. However, the undesirable correlations between the embryonic survivability and some analysed traits show the need to take into account all traits and their genetic correlations to achieve a balanced genetic progress. A new perspective for the implementation of genetic selection for E. faecalis susceptibility in laying hens is offered in the present dissertation. Practical implementation of selection against susceptibility to E. faecalis infection will require that breeding company is convinced that AA is of significant economic importance, which depends on disease incidence, production costs and losses associated with sick and/or dead birds, in addition to welfare and food safety concerns of consumers.