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Out of the 14 avian β-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian β-defensin 11 (Gga-AvBD11). Given its specific localization and its established antibacterial activity, Gga-AvBD11 appears to play a protective role in embryonic development. Gga-AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to β-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed β-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-β-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga-AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella, and avian influenza virus. Gga-AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga-AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.

Background Enterococcus cecorum (EC) infection currently is one of the most important bacterial diseases of modern broiler chickens but can also affect ducks or other avian species. However, little is known concerning pathogenesis of EC and most studies concentrate on examinations of EC strains from broilers only. The objective of this study was to compare pathogenic and commensal EC strains from different animal species concerning different phenotypic and genotypic traits. Results Pathogenic and commensal EC strains were not clearly separated from each other in a phylogenetic tree based on partial sequences of the 16S-rRNA-gene and also based on the fatty acid profile determined with gas chromatography. C 12:0 , C 14:0 , C 15:0 , C 16:0 , C 17:0 , C 18:0 , C 18:1 w7c, C 18:1 w9c and C 20:4 w6,9,12,15c were detected as the major fatty acids. None of the 21 pathogenic EC strains was able to utilize mannitol, while 9 of 29 commensal strains were mannitol positive. In a dendrogram based on MALDI-TOF MS data, pathogenic strains were not clearly separated from commensal isolates. However, significant differences concerning the prevalence of several mass peaks were confirmed between the two groups. Two different antisera were produced but none of the serotypes was predominantly found in the pathogenic or commensal EC isolates. Enterococcal virulence factors gelE , esp , asa1 , ccf , hyl and efaAfs were only detected in single isolates via PCR. No virulence factor was found significantly more often in the pathogenic isolates. The chicken embryo lethality of the examined EC isolates varied from 0 up to 100%. The mean embryo lethality in the pathogenic EC isolates was 39.7%, which was significantly higher than the lethality of the commensal strains, which was 18.9%. Additionally, five of the commensal isolates showed small colony variant growth, which was never reported for EC before. Conclusions Pathogenic and commensal EC isolates from different animal species varied in chicken embryo lethality, in their ability to metabolize mannitol and probably showed divergent mass peak patterns with MALDI-TOF MS. These differences may be explained by a separate evolution of pathogenic EC isolates. Furthermore, different serotypes of EC were demonstrated for the first time.

Campylobacter is an important human pathogen, and consumption of undercooked poultry has been linked to significant human illnesses. To reduce human illness, intervention strategies targeting Campylobacter reduction in poultry are in development. For more than a decade, there has been an ongoing national and international controversy about whether Campylobacter can pass from one generation of poultry to the next via the fertile egg. We recognize that there are numerous sources of Campylobacter entry into flocks of commercial poultry (including egg transmission), yet the environment is often cited as the only source. There has been an abundance of published research globally that refutes this contention, and this article lists and discusses many of them, along with other studies that support environment as the sole or primary source. One must remember that egg passage can mean more than vertical, transovarian transmission. Fecal bacteria, including Campylobacter, can contaminate the shell, shell membranes, and albumen of freshly laid fertile eggs. This contamination is drawn through the shell by temperature differential, aided by the presence of moisture (the \"sweating\" of the egg); then, when the chick emerges from the egg, it can ingest bacteria such as Campylobacter, become colonized, and spread this contamination to flock mates in the grow house. Improvements in cultural laboratory methods continue to advance our knowledge of the ecology of Campylobacter, and in the not-so-distant future, egg passage will not be a subject continuously debated but will be embraced, thus allowing the development and implementation of more effective intervention strategies.

Colibacillosis caused by Escherichia coli infections account for significant morbidity and mortality in the poultry industry. Yet, despite the importance of colibacillosis, much about the virulence mechanisms employed by avian E. coli remains unknown. In recent years several genes have been linked to avian E. coli virulence, many of which reside on a large transmissible plasmid. In the present study, a multiplex polymerase chain reaction (PCR) protocol to detect the presence of four of these genes is described. Such a protocol may supplement current diagnostic schemes and provide a rapid means of characterizing the E. coli causing disease in poultry. The targets of this procedure included iss, the increased serum survival gene; tsh, the temperature sensitive hemagglutinin gene; cvi, the ColV immunity gene; and iucC, a gene of the aerobactin operon. Organisms, known for their possession or lack of these genes, were used as a source of the template DNA to develop the multiplex PCR protocol. Identity of the amplicons was confirmed by size, DNA:DNA hybridization with specific gene probes, and DNA sequencing. When the multiplex PCR protocol was used to characterize 10 E. coli isolates incriminated in avian colibacillosis and 10 from the feces of apparently healthy birds, nine of the isolates from apparently healthy birds contained no more than one gene, while the 10th contained all four. Also, eight of the isolates incriminated in colibacillosis contained three or more genes, while the remaining two contained two of the target genes. Interestingly, the isolates of sick birds containing only two of the targeted genes killed the least number of embryos, and the isolate of healthy birds that contained all the genes killed the most embryos among this group. These genes were not found among the non–E. coli isolates tested, demonstrating the procedure's specificity for E. coli. Overall, these results suggest that this protocol might be useful in characterization and study of avian E. coli.

Escherichia coli is a common avian pathogen mainly associated with extraintestinal infections such as yolk sac infection (YSI). The aim of this study was to determine the serotypes and the presence of some virulence genes of E. coli strains isolated from different samples in a vertically integrated poultry operation in Mexico. Two hundred sixty-seven E. coli isolates from different samples were serotyped using rabbit serum against the 175 somatic (O) and 56 flagellar (H) antigens of the typing schema. Virulence genes were determined by colony blot hybridization, using DNA probes for st, eae, agg1, agg2, bfp, lt, cdt, slt, and ipaH diarrhea-associated virulence factors. The serogroup of 85% of the strains was determined; O19 (12%), O84 (9%), O8 (6%), and O78 (5%) were the most common. Using the complete antigenic formula (O and H), O19:NM (n = 31) was the serotype most frequently isolated from dead-in-shell embryos and in broilers that had died on the fourth, fifth, sixth, and seventh days after hatch. One hundred ten strains (41.2%) hybridized with one or more of the used probes. Of these, ipaH (72%), eae (30%), and cdt (27%) were the most common. Considering the origin of the respective isolates, 40% of the broiler farm strains were positive for at least one probe. Results show that some avian E. coli strains isolated in Mexico are included in avian pathogenic E. coli serotypes not previously reported, suggesting that they could be specific for this geographic area. The wide distribution of the ipaH gene among nonmotile strains suggests that this invasiveness trait could be important in YSI pathogenesis. On the other hand, some other genes could contribute to E. coli virulence during YSI.

In previous studies, the embryo lethality assay (ELA) was able to discriminate between virulent and avirulent avian Escherichia coli isolates and to predict percent mortality of the embryos resulting from an isolate based on three traits. The abilities to resist host complement, presence of Colicin V activity, and presence of the increased serum survival gene cluster (iss), were used together in a logistic regression analysis to predict the percentage of embryo deaths resulting from each of 20 avian E. coli isolates used in the ELA. In the present study, the same 20 isolates are used in an intravenous chicken challenge model in an effort to determine whether the ELA could be used to replace chicken challenge studies. Correlations between the mortality and a combination of mortality and morbidity (the survivors at trial termination with lesions suggestive of colibacillosis) and the previous ELA results and with selected isolate traits were performed. Additionally, resulting body weights in surviving chickens were compared between groups. The highest positive correlations were observed between the ELA and the combined mortality/morbidity of the intravenous challenge (r = 0.861, P < 0.0001 for the first ELA challenge, and r = 0.830, P < 0.0001 for the second ELA challenge). The IV challenge combined mortality/morbidity results had the highest correlation coefficients with the presence of iss (r = 0.864, P < 0.0001) and the expression of ColV (r = 0.878, P < 0.0001). The presence of tsh was slightly correlated with mortality (r = 0.465, P = .0389) but demonstrated a higher correlation with the combined mortality and morbidity of the IV challenge (r = 0.558, P = 0.0106). Even though the ELA results in a higher number of nonspecific deaths, the two challenge methods exhibit similar results and a high correlation with each other. Interestingly, some of the isolates showed differences in their ability to cause mortality between the ELA and the IV challenge model. Furthermore, some isolates reflected significant differences in body weights of surviving birds at IV trial termination.

Differentiating between virulent and avirulent avian Escherichia coli isolates continues to be a problem for poultry diagnostic laboratories and the study of colibacillosis in poultry. The ability of a laboratory to conduct one simple test that correlates with virulence would simplify studies in these areas; however, previous studies have not enabled researchers to establish such a test. In this study, the occurrence of certain phenotypic and genotypic traits purported to contribute to avian E. coli virulence in 20 avian E. coli isolates was correlated with the results of embryo challenge studies. This analysis was undertaken in an effort to determine which trait(s) best identified each avian E. coli isolate as virulent or avirulent. Traits selected were complement resistance, production of colicin V (ColV), motility, type F1 pili expression, presence of the temperature-sensitive hemagglutinin gene (tsh), and presence of the increased serum survival genetic locus (iss). ColV production, complement resistance, and presence of the iss genetic element were the three traits most highly correlated with high embryo lethality. A logistic regression model was used to predict the embryo lethality results on the basis of the most frequent isolate characteristics. Results indicate that ColV, complement resistance, and iss are significant predictor variables for the percentage of embryo lethality resulting from challenge with a specific avian E. coli isolate. However, no single trait has the ability to predict virulent isolates 100% of the time. Such results suggest the possibility that the embryo lethality assay may prove to be the one test needed to determine if an avian E. coli isolate is virulent.

Horizontal spread of Salmonella during hatching of broiler chicks was studied in three experiments. In each experiment, 120 unincubated, fertile hatching eggs were inoculated by immersion for 15 min in a 16 C physiological saline solution containing 1 X 10 colony forming units per ml of a nalidixic-acid-resistant strain of S. typhimurium. When inoculated eggs were transferred to hatchers after 17 to 18 days of incubation, control eggs at the same stage of incubation were added to the same tray and to trays above and below the tray containing inoculated eggs. Fertile inoculated eggs hatched at a rate of 86%, despite the high level of Salmonella contamination, indicating that chicks in eggs contaminated with salmonellae are likely to hatch and may contaminate other chicks in the same hatcher cabinet. Air samples showed a sharp increase in contamination in the hatcher at 20 days of incubation. Approximately 58% of mouth swabs and 90% of chick rinses were Salmonella-positive, in both inoculated and control eggs. In samples from inoculated eggs, Salmonellawas detected in the digestive tract of 8% of embryos at transfer from incubator to hatcher and in 55% of chicks at hatch. From control eggs, 44% of digestive tracts of hatched chicks were positive, indicating that Salmonella in a contaminated hatcher can reach the gut of chicks hatching from Salmonella-free eggs before they are removed from the hatcher

Mycoplasma meleagridis (MM) is a known pathogen for turkeys only. In this study, MM was used to inoculate chicken embryos and tumor cells to assess its pathogenic potential for chickens. In chicken embryos, it caused abnormal-shaped toes and severely denuded tracheae. In chicken tumor cells, MM reduced the cellular capacity to release a chemoattractant that causes the migration of heterophils. MM also caused death and/or a reduced growth rate in chicken HD-11 cells, a macrophage-monocyte-derived cell line. Thus, the data show that MM is a potential pathogen for chicken embryos and chickens cells. Further exploration to determine the pathogenicity in chickens may be warranted. /// El Mycoplasma meleagridis es agente patógeno exclusivo de los pavos. Se empleó el M. meleagridis para inocular embriones de pollo y células tumorales con el fin de evaluar su patogenicidad potencial en pollos. En embriones de pollo, el M. meleagridis ocasionó anormalidades en los dedos y tráqueas sin epitelio. En células tumorales de pollo, el M. meleagridis redujo la capacidad celular de liberar un agente quimioatrayente que ocasiona la migración de heterófilos. El M. meleagridis ocasionó igualmente la muerte y una reducción en el índice de crecimiento en las células de pollo HD-11, una línea celular derivada de macrófagos y monocitos. Los datos demuestran que el M. meleagridis es un patógeno potencial para embriones de pollo y células de pollo. Es necesario realizar estudios adicionales para determinar la patogenicidad del M. meleagridis en pollos.

The aim of this study was to evaluate the practicability of the chick embryo chorioallantoic membrane (CAM) with special regard to the ‘natural air sac' technique (NAST) of preparation for in-vivo research on the invasive potential of bacterial strains of various enterobacterial species. It was sought to establish an experimental system more closely resembling in-vivo conditions than cell lines on one hand, and cheaper and easier to handle than established animal models on the other.Fertilized eggs of the domestic fowl were incubated. The CAM was prepared atraumatically at the natural air space of the egg, and a cannula was inserted for subsequent extraction of allantoic fluid (AF) below the CAM. The CAM was then inoculated with either one out of five strains of Klebsiella pneumoniae an Escherichia coli K-12 strain or a Salmonella typhimurium strain, either alone or in combinations, respectively. AF samples were extracted at certain time points, and the presence of bacteria was determined by cultivation. Penetration and mortality ratios of the infected embryos were calculated. In addition, the mode of crossing the epithelial barrier was examined by electron microscopy.Differing rates of invasion through the CAM and rates of mortality of the chicken embryos demonstrated a clear dependency on the inoculated bacterial strain. Low invading bacteria could be distinguished from intermediate strains, and from strains exerting a strong capability of invasion and killing of the embryos. Simultaneous monotopical inoculation of Klebsiella and E. coli showed a permissive effect of co-incubated Klebsiella on the invasiveness of E. coli.The chick embryo CAM prepared by NAST has shown to be a useful model for in vivo studies on invasion capabilities, pathogenicity and interactions of inoculated bacteria.