Structure, function, and evolution of Gga-AvBD11, the archetype of the structural avian-double-ß-defensin family.

Out of the 14 avian ß-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian ß-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 ß-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed ß-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-ß-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.

Transcriptome modulation by in ovo delivered Lactobacillus synbiotics in a range of chicken tissues.

Synbiotics are the bioactive compounds that synergistically combine effects of prebiotics and probiotics. In poultry, synbiotics can be used to reprogram animal's intestinal microbiota upon perinatal in ovo injection on day 12 of eggs incubation. Optimally composed synbiotic delivered in ovo efficiently stimulates the host's intestinal microflora, which in turn exerts beneficial effects on the host and improves its physiological functions. The aim of the study was to estimate long-term changes in the chicken transcriptome after a single in ovo administration of two different synbiotics. On day 12 of eggs incubation, 5850 eggs of broiler chicken were distributed to experimental groups and injected with synbiotic 1 (S1)- Lactobacillus salivarius with galactooligosaccharides (GOS) or synbiotic 2 (S2)- Lactobacillus plantarum with raffinose family oligosaccharides (RFO). On day 21 post-hatching cockerels were sacrificed and immunological (cecal tonsils and spleen), intestinal (jejunum) and metabolic (liver) tissues were collected (n = 5). Isolated RNA served as a template for the whole-transcriptome analysis using GeneChip Chicken Gene 1.1. ST Array Strip (Affymetrix). Data analysis was performed using Affymetrix Expression Console and Transcriptome Analysis Console software, Venn diagrams, DAVID and CateGOrizer. The highest number of Differentially Expressed Genes (DEG) was detected in cecal tonsils (160 DEG) after S1 in ovo injection, and in liver (159 DEG) after S2 injection. The influence of S1 on transcriptome modulation was demonstrated by a strong activation of the genes taking part in the pathways related to metabolism and immune response in cecal tonsils. S2 injection led to modulation of the gene expression associated with metabolic and developmental signaling pathways in the liver. Obtained results let us conclude that synbiotics delivered in ovo have significant impact on chicken transcriptome and their effect depends on the composition of the bioactive compound.

Evaluating bacterial colonization of a developing broiler embryo after in ovo injection with a bioluminescent bacteria.

In ovo injection of probiotics has been of interest for achieving early health benefits. However, there is limited research demonstrating where bacteria could migrate within the embryo after injection. The objective of this study was to evaluate bacterial colonization or migration after in ovo injection of broiler embryo with bioluminescent Escherichia coli. Injection using 106 CFU/mL nonpathogenic E. coli was applied to amniotic and air cell regions on day 18 of incubation. On days 18, 19, 20, and 21 the amnion, skin, lung, gastrointestinal tract (GIT), bursa, and spleen were collected. On day 21, the GIT was separated into crop, duodenum, jejunum, ileum, and ceca sections. All tissues were visualized using anin vivo imaging system to confirm the presence of bioluminescent E. coli. Samples were homogenized, 10-fold serially diluted, and spread onto appropriate agar to determine bacterial loads in all tissues. Results indicated that eggs injected into the amnion had significantly high numbers of E. coli cells in all tissues compared to air cell injected and control treatments 2 h post-injection (P < 0.0001). E. coli was also found on the lungs, spleen, and bursa of eggs injected either in the amnion or air cell (P < 0.05). Results indicated that in ovo injection into the amnion was more efficient than air cell injection, yielding a higher bacterial concentration in the evaluated tissues, specifically the ileum and ceca. Future research using bioluminescent probiotic bacteria may establish sites of preference for different probiotics leading to site-specific application that can maximize their overall impact when in ovo injected.

Investigating commercial in ovo technology as a strategy for introducing probiotic bacteria to broiler embryos.

Probiotics can improve broiler performance and reduce pathogens. Because the hatchery can be a source of contamination, delivering probiotics to the embryo before hatch is desirable. To date, probiotics have primarily been injected into eggs manually. Therefore, the objective of this study was to deliver various probiotic bacteria into broiler hatching eggs using an automated commercial in ovo injection system to evaluate hatchability of fertile eggs (HF). Three separate experiments were conducted using Lactobacillus acidophilus, Bacillus subtilis, or Bifidobacterium animalis. In each experiment, 7 treatments (non-injected control; dry punch control; diluent-injected control; and injections of 103 cfu, 104 cfu, 105 cfu, or 106 cfu of bacteria/50 µL of diluent) were evaluated using 10 replicates per treatment. For each experiment, 2,490 eggs were obtained from a commercial hatchery. Eggs were incubated under standard incubation conditions. At 10 d of incubation (doi), eggs were candled, and infertile eggs were removed. On 18 doi, all eggs were injected with the appropriate treatment using an automated in ovo injection system. Once all eggs were injected, they were transferred to hatching baskets and placed into the hatcher. On 21 doi, chicks were removed from the hatcher, counted, and weighed. Hatch residue analysis was conducted to determine infertile, early dead, mid dead, late dead, pipped, cracked, contaminated, and cull chick statuses of all unhatched eggs. Injecting L. acidophilus, even at a concentration as high as 106 cfu/50 µL, did not impact hatch residue analysis (P > 0.05). However, HF was significantly less for eggs treated with B. subtilis than for control eggs (P < 0.0001). For the non-injected control, HF was 91%, but as concentration of B. subtilis increased, HF decreased to as low as 1.67% for the 105 cfu treatment. Late deads, pipped, and contaminated egg percentages were higher, and chick BW was lower for B. subtilis treatment groups compared to controls. In conclusion, L. acidophilus and B. animalis but not B. subtilis, appear to be suitable candidates for in ovo injection as probiotics.

Chicken embryo lethality assay for determining the lethal dose and virulence of Enterococcus faecalis.

Enterococcus faecalis is the major pathogen found in field cases of amyloid arthropathy in chickens. Given the need for a better understanding of the virulence mechanisms of the causative strains, the embryo lethality assay (ELA) is proposed in the present study as a model to evaluate the virulence of E. faecalis strains, specifically the pathogenic avian strain K923/96, which was previously related with amyloid arthropathy. Hence, 0.2 ml of five doses of the cited strain (from 2.5 to 2500 colony-forming units (CFU) per ml) were inoculated into the allantoic cavity 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 median lethal dose (LD50) was calculated. The ELA was repeated four times on a sample of 3443 eggs. 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. As expected, the higher the infectious dose, the greater the EMR and the lower the embryo survival time. The highest EMR was recorded after three and four days post-inoculation in all doses. In conclusion, these results can be used as a basis for further researches on the E. faecalis virulence. In order to corroborate its model capacity to predict the virulence of this bacterium, more ELAs with different E. faecalis strains are required.

O-polysaccharide is important for Salmonella Pullorum survival in egg albumen, and virulence and colonization in chicken embryos.

The pathogen Salmonella Pullorum is the causative agent of persistent systemic infection of poultry, leading to economic losses in developing countries due to morbidity, mortality and reduction in egg production. These infections may result in vertical transmission to eggs or progeny. Limited information is available regarding the mechanisms involved in the survival of Salmonella Pullorum in egg albumen and developing chicken embryos. Hence, we investigated the role of O-polysaccharide in the contamination of eggs and the colonization of chicken embryos. Compared with the wild-type strain, the isogenic waaL mutant exhibited an O-antigen-deficient rough phenotype, and increased sensitivity to egg albumen and chicken serum, as well as reduced adherence to DF-1 cells. Infection with Salmonella Pullorum lacking O-polysaccharide resulted in significantly reduced embryo lethality and bacterial colonization. These results suggest that O-polysaccharide is essential for Salmonella Pullorum colonization in eggs, both post-lay and developing embryos. The chicken embryo infection model could be used to characterize the interaction between Salmonella Pullorum and developing embryos, and it will also contribute to the development of more rational vaccines to protect laying hens and embryos.

Comparison of pathogenic and non-pathogenic Enterococcus cecorum strains from different animal species.

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. C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C18:1 w7c, C18:1 w9c and C20:4 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.

Prevalence, Virulence Potential, and Antibiotic Susceptibility Profile of Listeria monocytogenes Isolated From Bovine Raw Milk Samples Obtained From Rajasthan, India.

Listeriosis is a serious foodborne disease of a global concern, and can effectively be controlled by a continuous surveillance of the virulent and multidrug-resistant strains of Listeria monocytogenes. This study was planned to investigate prevalence of L. monocytogenes in bovine raw milk samples. A total of 457 raw milk samples collected from 15 major cities in Rajasthan, India, were analyzed for the presence of L. monocytogenes by using standard microbiological and molecular methods. Five of the 457 samples screen tested positive for L. monocytogenes. Multiplex serotyping showed that 3/5 strains belonged to serotype 4b followed by one strain each to 1/2a and to 1/2c. Further virulence potential assessment indicated that all strains possessed inlA and inlC internalins, and, in addition, two strains also possessed the gene for inlB. All strains were positive for Listeriolysin O (LLO) and showed phosphatidylinositol-specific phospholipase C (PI-PLC) activity on an in vitro agar medium with variations in production levels among the strains. A good correlation between the in vitro pathogenicity test and the chick embryo test was observed, as the strains showing higher LLO and PI-PLC activity were found to be lethal to fertilized chick embryos. All strains were resistant to the majority of antibiotics and were designated as multidrug-resistant strains. However, these strains were susceptible to 9 of the 22 tested antibiotics. The maximum zone of inhibition (mm) and acceptable minimum inhibitory concentration were observed with azithromycin, and thus it could be the first choice of a treatment. Overall, the presence of multidrug-resistant L. monocytogenes strains in the raw milk of Rajasthan region is an indicator of public health hazard and highlighting the need of consumer awareness in place and implementation of stricter food safety regulations at all levels of milk production.

Ileal MUC2 gene expression and microbial population, but not growth performance and immune response, are influenced by in ovo injection of probiotics in broiler chickens.

1. The objective of present study was to evaluate the effects of intra-amniotic injection of different probiotic strains (Bacillus subtilis, Enterococcus faecium and Pediococcus acidilactici) on the intestinal MUC2 gene expression, microbial population, growth performance and immune response in broiler chicken. 2. In a completely randomised design, different probiotic strains were injected into the amniotic fluid of the 480 live embryos (d 18 of incubation), with 4 treatments and 5 replicates. Ileal MUC2 gene expression, microbial profile, growth performance and immune response were determined. 3. Injection of probiotic strains, especially B. subtilis, had significant effect on expression of the MUC2 on d 21 of incubation and d 3 post-hatch, but not on d 19 of incubation. 4. Injection of the probiotic strains decreased significantly the Escherichia coli population and increased the lactic acid bacteria population during the first week post-hatch. 5. Inoculation of probiotics had no significant effect on antibody titres against Newcastle disease virus, antibody titres against sheep red blood cell and cell-mediated immune response of chickens compared to control. 6. In ovo injection of the probiotic strains had no significant effect on growth performance of broiler chickens. 7. It was concluded that injection of probiotic bacteria especially B. subtilis into the amniotic fluid has a beneficial effect on ileal MUC2 gene expression and bacteria population during the first week post-hatch, but has no effect on growth performance and immune response in broiler chickens.

Characterization of egg white antibacterial properties during the first half of incubation: A comparative study between embryonated and unfertilized eggs.

Egg white is an important contributor to the protection of eggs against bacterial contaminations during the first half of incubation (day zero to 12), prior to the egg white transfer into the amniotic fluid to be orally absorbed by the embryo. This protective system relies on an arsenal of antimicrobial proteins and on intrinsic physicochemical properties that are generally unfavorable for bacterial multiplication and dissemination. Some changes in these parameters can be observed in egg white during egg storage and incubation. The aim of this work was to characterize changes in the antibacterial potential of egg white in embryonated eggs (FE) during the first half of incubation using unfertilized eggs (UF) as controls. Egg white samples were collected at day zero, 4, 8, and 12 and analyzed for pH, protein concentration, and protein profile. Antibacterial properties of egg white proteins were evaluated against Listeria monocytogenes, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, and Salmonella Enteritidis. During incubation, differential variations of egg white pH and protein concentrations were observed between UF and FE. At equal protein concentrations, similar activities against L. monocytogenes and S. uberis were observed for FE and UF egg white proteins. A progressive decline in these activities, however, was observed over incubation time, regardless of the egg group (UF or FE). SDS-PAGE analysis of egg white proteins during incubation revealed discrete changes in the profile of major proteins, whereas the stability of some less abundant antimicrobial proteins seemed more affected. To conclude, the antibacterial activity of egg white proteins progressively decreased during the first half of egg incubation, possibly resulting from the alteration of specific antimicrobial proteins. This apparent decline may be partly counterbalanced in embryonated eggs by the increase in egg white protein concentration. The antibacterial potential of egg white is very effective during early stages of embryonic development but its alteration during incubation suggests that extra-embryonic structures could then progressively ensure protective functions.

Experimental infection of chicken embryos with recently described Brucella microti: Pathogenicity and pathological findings.

Brucellae are facultative intracellular pathogens causing disease in a wide range of domestic and wild animals as well as in humans. Brucella (B.) microti is a recently recognized species and was isolated from common voles (Microtus arvalis), red foxes and soil in Austria and the Czech Republic. Its pathogenicity for livestock and its zoonotic potential has not been confirmed yet. In the present study 25 SPF chicken embryos were inoculated at day 11 of age with 1.6×10(3) and 1.6×10(5)B. microti by yolk sac and allantoic sac routes. Re-isolation of B. microti indicated rapid multiplication of bacteria (up to 1.7×10(12)CFU). B. microti provoked marked gross lesions, i.e. hemorrhages and necroses. All inoculated embryos were dead (100% mortality) in between 2nd and 4th day post inoculation. The predominant histopathological lesion was necroses in liver, kidneys, lungs, spleen, gastrointestinal tract, spinal meninges, yolk sac and chorioallantoic membrane. Immunohistochemical examination showed the presence of Brucella antigen in nearly all of these organs, with infection being mainly restricted to non-epithelial cells or tissues. This study provides the first results on the multiplication and pathogenicity of the mouse pathogenic B. microti in chicken embryos. These data suggest that, even though chicken are not mammals, they could provide a useful tool for understanding the pathogenesis of B. microti associated disease.

Csr1/Zap1 Maintains Zinc Homeostasis and Influences Virulence in Candida dubliniensis but Is Not Coupled to Morphogenesis.

The supply and intracellular homeostasis of trace metals are essential for every living organism. Therefore, the struggle for micronutrients between a pathogen and its host is an important determinant in the infection process. In this work, we focus on the acquisition of zinc by Candida dubliniensis, an emerging pathogen closely related to Candida albicans. We show that the transcription factor Csr1 is essential for C. dubliniensis to regulate zinc uptake mechanisms under zinc limitation: it governs the expression of the zinc transporter genes ZRT1, ZRT2, and ZRT3 and of the zincophore gene PRA1. Exclusively, artificial overexpression of ZRT2 partially rescued the growth defect of a csr1Δ/Δ mutant in a zinc-restricted environment. Importantly, we found that, in contrast to what is seen in C. albicans, Csr1 (also called Zap1) is not a major regulator of dimorphism in C. dubliniensis. However, although a csr1Δ/Δ strain showed normal germ tube formation, we detected a clear attenuation in virulence using an embryonated chicken egg infection model. We conclude that, unlike in C. albicans, Csr1 seems to be a virulence factor of C. dubliniensis that is not coupled to filamentation but is strongly linked to zinc acquisition during pathogenesis.

Effects of synbiotics injected in ovo on regulation of immune-related gene expression in adult chickens.

OBJECTIVE: To determine immunomodulatory effects of synbiotics administered in ovo on immune-related gene expression in adult chickens. ANIMALS: 30 Green-legged Partridgelike chickens. PROCEDURES: On incubation day 12, eggs were injected with 3 synbiotics (Lactococcus lactis subsp lactis IBB SL1 with raffinose family oligosaccharides [RFOs; S1], Lactococcus lactis subsp cremoris IBB SC1 with RFOs [S2], and Lactobacillus acidophilus and Streptococcus faecium with lactose [S3]). Control eggs were injected with RFOs prebiotic or saline (0.9% NaCl) solution. Gene expression of 6 cytokines (interleukin [IL]-4, IL-6, IL-12p40, IL-18, interferon [IFN]-ß, and IFN-γ) and 1 chemokine (IL-8) was analyzed in the cecal tonsils and spleen of 6-week-old chickens by means of reverse transcription quantitative PCR assays. RESULTS: Gene expression for IL-4, IL-6, IFN-ß, and IL-18 was significantly upregulated in the spleen of chickens in groups S2 and S3. In contrast, IL-12 expression was downregulated in group S2 and IFN-γ expression was downregulated in group S3. Expression of IL-8 did not change in chickens treated with synbiotics in ovo. Gene expression of all cytokines, except for IL-18, was downregulated in cecal tonsils. CONCLUSIONS AND CLINICAL RELEVANCE: In ovo administration of synbiotics activated the immune system in adult chickens. The intestinal immune system (cecal tonsils) had downregulation of expression for the cytokines evaluated, which indicated an increase in oral tolerance, whereas in the peripheral part of the immune system (spleen), expression of IL-4 and IL-6 was upregulated. Evaluation of immune-related gene expression patterns may be useful when monitoring the effectiveness of synbiotic selection with respect to immunobiotic properties.

In ovo inoculation of chicken embryos with probiotic bacteria and its effect on posthatch Salmonella susceptibility.

The feasibility of establishing probiotic bacteria in the intestine of broiler chickens by in ovo inoculation was investigated, followed by verifying possible subsequent protection against Salmonella Enteriditis infection. In a first study, 7 commercially available probiotics were screened for compatibility with in ovo inoculation. Two of these probiotics, one being a Enterococcus faecium and the other a Bacillus subtilis, were selected for colonizing the chick gut without compromising hatchability. In a second study, these 2 products were administered in ovo and in the feed to chicks reared until 18 d in comparison with noninoculated chicks and with chicks fed an antibiotic. All chicks were orally challenged with Salmonella Enteritidis at 4 d of age. Results showed reduced performance of Salmonella Enteritidis challenged chicks fed no additives compared with challenged chicks fed antibiotic, but no significant differences in mortality was observed. Probiotics offered in ovo or through the diet could only partially recover performance compared with antibiotic-fed chicks. A significant reduction in the number of Salmonella Enteritidis positive chicks was observed when chicks were in ovo inoculated with E. faecium and continued receiving it in the diet. This work establishes standards for future in ovo colonization research and emphasizes its value as a promising method to deliver individual precise dose of probiotics to poultry in mass scale at the earliest possible age based on the competitive exclusion concept. In ovo colonization with probiotic can therefore become an important ally in combination with other approaches to combat Salmonella and other intestinal bacterial infections in poultry.

Effect of immersion and inoculation in ovo of Lactobacillus spp. in embryonated chicken eggs in the prevention of Salmonella Enteritidis after hatch.

The protection level against Salmonella Enteritidis was evaluated in chickens after in ovo treatment with different species of Lactobacillus spp. inoculated into the air cell or by immersion in broth culture. Two hundred forty embryonated eggs were distributed into 8 groups, corresponding to treatments with Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus salivarius, and control. On d 18 of incubation, 4 groups were inoculated with 0.1 mL of inoculum in the air cell and 4 groups were immersed for 3 min in culture of each treatment. Two days after hatching, 0.5 mL of Salmonella Enteritidis culture was inoculated by the intraesophageal route. On d 5 of life, the chicks were euthanized and the ceca were processed to obtain Salmonella Enteritidis counts. There was no decrease in Salmonella Enteritidis colonization of chick ceca, regardless of treatment or route of administration. Lactobacillus spp. samples used in the treatment showed no probiotic potential in chicks when inoculated in ovo, in relation to Salmonella Enteritidis inhibition in poultry ceca.

Polymerase chain reaction detection of naturally occurring Campylobacter in commercial broiler chicken embryos.

Campylobacter, a foodborne pathogen closely associated with poultry, is recognized as a leading bacterial etiologic agent of human gastroenteritis in the United States. In this investigation, 2 trials were performed where tissues from 7-, 14/15-, and 19-d-old commercial broiler chicken embryos were tested for the presence of Campylobacter using both culturing methodology and PCR. Conventional culturing methods failed to detect Campylobacter from any samples tested during this investigation. Using a set of primers specific for the Campylobacter flagellinA short variable region (flaA SVR), Campylobacter DNA was amplified in 100, 80, and 100% of gastrointestinal tracts from 7-, 15-, and 19-d-old embryos, respectively, in the first trial. Similarly, Campylobacter DNA was detected in 100, 70, and 60% of gastrointestinal tracts of 7-, 14-, and 18-d-old embryos, respectively, in the second trial. In both trials, yolk sac, albumin, and liver/gallbladder samples from 19-d-old embryos all failed to produce amplicons indicative of Campylobacter DNA. Subsequent DNA sequence analyses of the flaA SVR PCR products were consistent with the amplicon arising from Campylobacter. Although a determination of whether the Campylobacter was living or dead within the embryos could not be made, these results demonstrate that Campylobacter-specific DNA is present within the gastrointestinal tract of broiler chicken embryos; however, the means by which it is present and the relative contribution to subsequent Campylobacter contamination of poultry flocks requires further investigation.

Evidence for horizontal and vertical transmission in Campylobacter passage from hen to her progeny.

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.

Embryonated chicken eggs as alternative infection model for pathogenic fungi.

Embryonated eggs have been used as infection models for decades in virology and bacteriology. However, they can also be used as an attractive alternative infection model for studying fungal pathogenesis. Here, we discuss some general aspects which need to be considered when working with embryonated eggs as infection models. Furthermore, we provide detailed protocols and technical tips for infection of embryonated eggs with Aspergillus fumigatus and Candida albicans via the chorioallantois membrane, as well as sampling methods for downstream analyses.

The embryo lethality of Escherichia coli isolates and its relationship to the presence of virulence-associated genes.

The aims of this study were to determine if the chicken embryo lethality assay and the presence of 9 virulence-associated genes of Escherichia coli were correlated and to discover which virulence genes contributed most to embryo lethality. We examined 58 E. coli strains isolated from visceral organs of chickens with colibacillosis for the presence of 9 virulence genes (fimC, tsh, fyuA, irp2, iucD, cvi/cva, iss, astA, and vat) by PCR. The gene FimC (type I fimbriae) was detected with the highest prevalence in 93.1% of the isolates, followed by iucD (67.24%), iss (58.62%), tsh (34.48%), cvi/cva (34.48%), fyuA (32.76%), astA (31.0%), irp2 (27.59%), and vat (17.24%). The embryo mortality ranged from 5 to 100%; however, most of the isolates were moderately or highly virulent. High positive correlations were observed between the presence of virulence genes and chicken embryo lethality. In addition, presence of the iucD (aerobactin) gene was the trait that best contributed to embryo mortality by using the multivariate model. These results suggest that expression frequency of these 9 virulence genes is associated with embryo mortality, and the gene that best predicted embryo mortality was iucD.

Transfer, viability and colonisation of Campylobacter jejuni in the chicken vitellus and in embryos.

1. The objective of this study was to evaluate the ability of Campylobacter jejuni to penetrate and colonise eggs from specific-pathogen-free (SPF) and heavy breeder hens, and to determine its effects on the viability of SPF embryos. 2. We detected C. jejuni in 10% of breeder hens and 20% of SPF eggs, which demonstrates the ability of the bacteria to go through the pores of eggs and contaminate the vitellus after 3 h of contact. These results indicate that there is a risk of contamination under commercial production conditions, where, after oviposition, there is contact between the egg and organic material such as faeces and blood. 3. We observed that in 80% of SPF eggs analysed, C. jejuni survived the 21-d incubation period. This positive result suggests that this microorganism was also responsible for early embryonic mortality. 4. The ability of C. jejuni to penetrate the eggs in this study suggests that serious problems may occur under natural field conditions, which may cause significant problems for producers.