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.

Multiplication of Salmonella enteritidis in egg yolks after inoculation outside, on, and inside vitelline membranes and storage at different temperatures.

Prompt refrigeration to restrict bacterial growth is important for reducing eggborne transmission of Salmonella enterica serovar Enteritidis (SE). The nutrient-rich yolk interior is a relatively infrequent location for initial SE deposition in eggs, but migration across the vitelline membrane can result in rapid bacterial multiplication during storage at warm temperatures. The objective of the present study was to measure the multiplication of SE in yolks after introduction at three different locations and subsequent storage at a range of temperatures. Using an in vitro egg contamination model, approximately 100 CFU of SE was inoculated either inside yolks, onto the exterior surface of vitelline membranes, or into the adjacent albumen. After storage of samples from each inoculation group at 10, 15, 20, and 25°C for 24 h, SE was enumerated in yolks. For all three inoculation locations, the final SE levels in yolks increased significantly with increasing storage temperatures. At all storage temperatures, significant differences in SE multiplication were observed between inoculation sites (yolk inoculation>vitelline membrane inoculation>albumen inoculation). At 25°C, final log concentrations of 7.759 CFU of SE per ml (yolk inoculation), 2.014 CFU/ml (vitelline membrane inoculation), and 0.757 CFU/ml (albumen inoculation) were attained in yolks after storage. These results demonstrate that, even when the initial site of SE deposition is outside the egg yolk, substantial multiplication supported by yolk nutrients can occur during the first day of storage and the risk of bacterial growth increases at higher ambient storage temperatures.

In vitro penetration of Salmonella Enteritidis through yolk membranes of eggs from 6 genetically distinct commercial lines of laying hens.

Although deposition of Salmonella Enteritidis inside yolks is less common than deposition in albumen or on the vitelline (yolk) membrane in naturally contaminated eggs laid by infected hens, bacterial migration into the yolk to reach its nutrient-rich contents could lead to extensive multiplication. The present study used an in vitro egg contamination model to assess the ability of small initial numbers of Salmonella Enteritidis to penetrate the vitelline membrane and multiply inside yolks of eggs laid by 6 genetically distinct commercial lines of hens during 24 h of storage at 30 degrees C. Eggs from each line were tested at 4 different hen ages by inoculation of approximately 100 cfu of Salmonella Enteritidis onto the outside of the vitelline membranes of intact yolks in plastic centrifuge tubes and then adding back the albumen into each tube before incubation. Overall, the frequency of penetration of Salmonella Enteritidis into the yolk contents of eggs from individual lines of hens ranged from 30 to 58% and the mean concentration of Salmonella Enteritidis in yolk contents after incubation ranged from 0.8 to 2.0 log(10) cfu/mL. For both of these parameters, values for one hen line were significantly higher than for 2 other lines, but no other differences were observed. Hen age did not have a significant effect on egg yolk penetration by Salmonella Enteritidis. These results indicate that opportunities for the migration and growth of small initial numbers of Salmonella Enteritidis to attain more dangerous levels inside contaminated eggs during storage at warm temperatures can sometimes vary between different lines of laying hens.

Multiplication of Salmonella enteritidis on the yolk membrane and penetration to the yolk contents at 30 degrees C in an in vitro egg contamination model.

Refrigeration to limit bacterial multiplication is a critical aspect of efforts to control the transmission of Salmonella enterica serovar Enteritidis (SE) to consumers of contaminated eggs. Although the nutrient-rich yolk interior is an uncommon location for SE contamination in freshly laid, naturally contaminated eggs, migration across the vitelline membrane could lead to rapid bacterial multiplication even when the initial site of deposition is outside the yolk. Multiplication on the yolk membrane (before, or in addition to, multiplication within the yolk contents) could be another source of increased risk to consumers. The present study used an in vitro egg contamination model to compare the abilities of four strains of SE to either multiply in association with the yolk membrane or migrate through that membrane to reach the yolk contents during 36 h of incubation at 30 degrees C. After inoculation onto the exterior surface of intact, whole yolks, all four SE strains penetrated the vitelline membrane to reach the yolk contents (at an overall frequency of 11.5%) after 12 h of incubation. The mean log concentration of SE was significantly higher in whole yolks (including yolk membranes) than in yolk contents at both 12 h (0.818 versus 0.167 CFU/ ml) and 36 h (2.767 versus 1.402 CFU/ml) of incubation. These results demonstrate that SE multiplication on the vitelline membrane may both precede and exceed multiplication resulting from penetration into the yolk contents during the first 36 h of unrefrigerated storage, reinforcing the importance of rapid refrigeration for protecting consumers from egg-transmitted illness.

In vitro study of Salmonella enteritidis and Salmonella typhimurium definitive type 104: survival in egg albumen and penetration through the vitelline membrane.

Salmonella enteritidis and Salmonella typhimurium definitive type 104 (DT104) have been detected in the chicken oviduct, and their survival in egg albumen at the chicken body temperature of 42 degrees C may be important in oviductal and transovarian contamination of intact shell eggs. Eight S. enteritidis and 24 S. typhimurium DT104 strains were tested for their in vitro survival in egg albumen. The concentration of the organisms declined more rapidly when incubated at 42 degrees C than at 37 degrees C and dropped to nondetectable levels within 96 h at the higher, but not at the lower, temperature. In another experiment, 3 S. enteritidis and 3 S. typhimurium DT104 strains were randomly selected, and dosages of 20 and 200 cells of each strain were inoculated onto the vitelline membranes of egg yolks, which were then submerged in the original albumen and incubated for 24 h at 42 degrees C. Under these conditions, the organisms survived in albumen but did not penetrate the vitelline membrane. However, in a similar experiment, penetration did occur when the specimens were incubated at 30 degrees C for 72 h. The results suggest that low numbers of S. enteritidis and S. typhimurium DT104 can be expected to survive in egg albumen during the 24-h period of egg formation in the oviduct but would be unlikely to invade the yolk.before oviposition. However, depending on storage conditions following oviposition, S. enteritidis, as well as S. typhimurium DT104, could survive longer and may eventually invade the egg yolks.

Individual-based modelling of growth and migration of Salmonella enteritidis in hens' eggs.

An individual-based model (IbM) was developed to describe the growth and migration of Salmonella enteritidis in hens' eggs. The Bacteria Simulator (BacSim) environment was used to implement the model; the bacteria are represented by spheres that grow by nutrient uptake and divide in two daughter cells upon exceeding a certain threshold volume. Motility of the Salmonella bacteria was described by a run and tumble mechanism. For the sake of simplicity, the bacteria were assumed to grow exponentially, an appropriate assumption for the initial phase of growth relevant for shelf-life predictions. Both albumen and yolk were assumed to be homogeneous. The impact of several model parameters (chemotaxis, growth rate, initial contamination numbers and bacterial swimming speed) was assessed by a sensitivity analysis. The results show that chemotaxis towards the yolk would have a strong effect on the time needed to reach the vitelline membrane, an aspect that future research should focus on. The contamination position had less impact on the time to reach the vitelline membrane. The simulation results illustrate the need for more detailed knowledge on the subject of bacterial migration in hens' eggs. Our model can easily incorporate this knowledge when it becomes available.

Contamination of egg shell and contents with Salmonella enteritidis: a review.

Salmonella enteritidis can contaminate the contents of clean, intact shell eggs as a result of infections of the reproductive tissue of laying hens. The principal site of infection would appear to be the upper oviduct. In egg contents the most important sites of contamination are either the outside of the vitelline membrane or the albumen surrounding it. In fresh eggs, only few salmonellas are present and as albumen is an iron-restricted environment, growth will only occur once storage-related changes to vitelline membrane permeability, which allow salmonellas to invade yolk contents, have taken place. When this happens high populations are achieved in both yolk contents and albumen. Some eggs from naturally infected hens have been found to contain large numbers of S. enteritidis. The rate of change in membrane permeability is temperature-dependent. In eggs stored at 20 degrees C, yolk invasion is uncommon until eggs have been stored for 3 weeks. In stimulated kitchen conditions where temperatures reached 30 degrees C, salmonellas could grow rapidly after a few days.

Egg age and the growth of Salmonella enteritidis PT4 in egg contents.

The growth of Salmonella enteritidis PT4 in albumen around an intact yolk was governed by the age of the egg on inoculation. In the majority of eggs, held at 20 degrees C, the bacterium was unable to grow rapidly until eggs had been stored for approximately 3 weeks. The multiplication of S. enteritidis in stored eggs appeared to be associated with alterations to the yolk membrane which allowed the bacterium to either invade the yolk or obtain nutrients from it. The rate at which egg contents change to permit the growth of S. enteritidis would appear to be temperature related and took place more rapidly when eggs were stored under conditions where temperatures fluctuated and, on occasions, reached 30 degrees C.

Gallus domestica yolk (vitelline) membrane use for in vitro microbial adherence studies.

The yolk membrane of unfertilized eggs (Gallus domestica) was isolated, washed, placed onto a glass holder, and utilized as an in vitro membrane model to study yeast attachment.

Ultrastructure of Legionella pneumophila.

Eleven lung samples positive for Legionnaires' disease, 12 strains of Legionella pneumophila cultured on various bacteriological media, and one strain growth in the yolk sac of fertile hens' eggs were examined by negative staining, thin sectioning, and scanning electron microscopy. All organisms studied were ultrastructurally similar irrespective of strain, source, or method of cultivation, presenting mainly as short rods, 0.6 x 1.5 micrometer, with tapered ends, though long forms and filaments were also evident. In this they resembled typical Gram-negative organisms. Division was by non-septate binary fission, and the cell wall was composed of two triple-unit membranes with morphological evidence of a peptidoglycan layer. The bacterial cytoplasm was rich in ribosomes and nuclear elements and often contained vacuoles. No acid polysaccharides or bacterial appendages were detected surrounding the organisms. In lung tissue and yolk sac membranes, the organisms replicated within the cytoplasm of infected cells and in the intercellular spaces and were specifically identified in thin sections by immunoferritin techniques.

Chicken embryo as an animal model for gonorrhea.

Parameters of infection of the chicken embryo with Neisseria gonorrhoeae were defined in order to standardize infectious and lethal doses. Virulent (T1) and avirulent (T3) gonococci from two strains were used to infect 7- to 12-day-old White Leghorn chicken embryos via the yolk sac (YS) or chorioallantoic membrane (CAM) route. Infection of embryos was established following YS inoculation of 1 to 10 viable gonococci. Although 8- to 10-day-old embryos were the most susceptible, an inoculum of less than 100 gonococci was sufficient to kill any age embryo via this route. Embryos were less susceptible to infection via the CAM, where an inoculum of from 10(5) to 10(6) colony-forming units was lethal by 42 h. Strain and morphological type had a variable influence on the ability of the gonococcus to infect and kill the chicken embryo by either route; however, agar-grown and broth-grown organisms produced consistently similar mean lethal dose (LD(50)) and mean infective dose (ID(50)) values. LD(50) and ID(50) differences between T1 and T3 gonococci from strain 72H641 were not apparent after either YS or CAM inoculation of 8- or 10-day chicken embryos, respectively. YS and CAM LD(50) values for strain 72H641 T1 and T3 and CDC 9 T3 were also similar; however, these values were slightly lower for CDC 9 T1. In terms of infectivity or colonization, CDC 9 T1 and T3 had higher ID(50) values via the YS and lower ID(50) values via the CAM than 72H641. CDC 9 T1 was slightly more infective via the YS and less infective via the CAM than its T3 counterpart. Although the gonococcal strain used will influence interpretation of results, infection of both YS and CAM was highly reproducible in terms of gross pathology and of LD(50) and ID(50) data for a particular strain and colony type.

Characterization of the Madrid E strain of Rickettsia prowazekii purified by renografin density gradient centrifugation.

The avirulent Madrid E strain of Rickettsia prowazekii cultivated in chicken yolk sacs could be purified successfully with a Renografin density gradient method developed previously for Rickettsia typhi. Recovery during purification, viability, and lack of contamination with host cell components were similar for the two species, although yields of R. prowazekii per yolk sac were lower. Purified typhus rickettsiae provided satisfactory antigens in the complement fixation, Ouchterlony double-diffusion, and microagglutination tests. The retention of the typhus soluble group antigen during purification was readily demonstrated by complement fixation tests. However, removal of the soluble group antigen by ether treatment was not always adequate for the demonstration of type-specific particulate antigens. Heat-killed R. prowazekii cells gave higher serum microagglutination titers than untreated or formalized cells, a difference was noted for R. typhi cells. Although the protein profiles of whole cells and extracts of R. typhi and R. prowazekii on sodium dodecyl sulfate-polyacrylamide gels were relatively similar, a small but reproducible, difference in the electrophoretic mobilities of their malate dehydrogenases was detected. Purification of typhus rickettsiae on Renografin gradients has no apparent adverse effects on their metabolic or antigenic properties.

Enzymatic activities leading to pyrimidine nucleotide biosynthesis from cell-free extracts of Rickettsia typhi.

Cell-free extracts from Rickettsia typhi were examined for the presence or absence of pyrimidine phosphotransferase enzymes and compared with the enzymes of mouse L cells and Salmonella typhimurium. The organisms were grown in mouse L cells and in the yolk sacs of chicken embryos, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The enzymes for the reutilization of uridine and thymidine, uridine kinase (EC 2.7.1.48) and thymidine kinase (EC 2.7.1.21), were not detected in R. typhi extracts with the phosphate donors effective for control enzymes. The following enzyme activities were demonstrated in R. typhi: uridine-5'-monophosphate kinase (UMPK, EC 2.7.4.4), deoxythymidine-5'-monophosphate kinase (dTMPK, EC 2.7.4.9), and nucleosidediphosphate kinase (NDPK, EC 2.7.4.6). Physicochemical and enzymatic analyses demonstrated that the pyrimidine nucleotide kinases of R. typhi were not of host origin and that the source (yolk sac and mouse L cells) did not influence the relative enzymatic activities. The specific activities of UMPK and dTMPK were higher when the rickettsiae were harvested before embryo death, whereas NDPK levels were slightly decreased. The specific activities of UMPK, dTMPK, and NDPK were comparable to those of S. typhimurium, and consequently the rickettsiae have potential for the anabolism of monophosphates, as do the host-independent bacteria. These results suggest that R. typhi cannot utilize host uridine or thymidine pools directly but must rely on themonophosphorylated molecules of the host cell or must synthesize the monophosphates de novo.

Electron microscopic study of virus particles in yolk sac and placenta and in hematopoietic organs of newborn C3Hf mice.

Examination of yolk sac from a C3Hf and a C3H mouse with the electron microscope revealed the presence of C-type virus particles in the blood islands. Particles were observed budding from the plasma membrane of hemocytoblasts, from erythroblasts, and occasionally from reticulocytes. C-type particles were also found in similar cells in hematopoietic foci in the liver, spleen, and bone marrow of embryos, and they continued to be present in newborn C3Hf mice up to 11 days of age. Particles consistently appeared in the thymus, even in older suckling mice. A comparison is made between the presence of C-type particles in organs of embryonic, newborn, and adult C3Hf mice. C-type particles were not observed in the chorioallantoic placentas from mice that were given injections of mouse leukemia virus (Gross) or from normal noninjected mice; however, intracisternal A-type particles were present in cytotrophoblast cells from these placentas.

[Cultivation of F. tularensis in developing chick embryos]. / Kultivirovanie F. tularensis v razvivaiushchikhsia kurinykh émbrionakh

In cultivation of the causative agent of tularemia in the organism of chick embryos (8-10 days of incubation) it was found that the virulent strains multiplied intensively in the yolk sacs causing the death of the embryos on the 3rd-4th day after the infection; as to the virulent strains-they were only preserved in the embryos. The period of death of the embryos and the accumulation of bacteria depended on the strain virulence. No differences in the anatomical structure of the cells belonging to the virulent and avirulent strains could be found.

Comparative infectivity of trachoma organisms in HeLa 229 cells and egg cultures.

Thirty-two trachoma strains were simultaneously titrated in the yolk sac of embryonated chicken eggs and in HeLa 229 cells pretreated with diethylaminoethyl-dextran to determine the relative sensitivity of the two culture systems for detection of the infection. The strains tested were both yolk sac- and HeLa cell-established isolates from the eye and the genital tract. The study showed that the cell culture was of equal or greater sensitivity than yolk sac culture and that ocular and genital isolates differ in their ability to propagate in egg and cell culture: genital strains grow better in cell culture than in eggs and ocular strains grow only slightly better in cell culture.

Ultrastructural studies of Chlamydia psittaci 6BC in situ in yolk sac explants and L cells: a comparison with gram-negative bacteria.

Chlamydia psittaci (6BC) was grown in yolk sac explants and in L cells and fixed by perfusion in situ to provide undamaged material for comparison with gram-negative bacteria. Reticulate, intermediate, and elementary bodies were all seen to lack a well-defined periplasmic space; intermediate and elementary bodies showed condensations of the nucleoid which differ from common bacterial configurations; and the cytoplasm of highly condensed elementary bodies was much more electron dense than that of the gram-negative bacteria, while retaining its basically particulate nature. These important morphological distinctions are interpreted as reflections of a significantly different cellular level of organization in these two groups of organisms. No important morphological differences were noted in comparisons of the chlamydial particles grown in the two different host systems.

The effect of purification on the ultrastructure and infectivity of egg-attenuated Chlamydia psittaci (6BC).

A procedure is described for the purification of mixed populations of the three different morphological forms of Chlamydia psittaci (6BC) from infected yolk sac membranes. Elementary bodies and small intermediate bodies are not perceptibly damaged during purification which involves homogenization of the host cells, differential centrifugation, sedimentation through 20% sucrose, and treatment with trypsin. The observation that elementary bodies undergo plasmolysis in 20% sucrose is interpreted as indicating that the cytoplasmic membrane of these particles is intact at that stage in the purification. Reticulate bodies and large intermediate bodies are damaged, to a degree, by the homogenization step. This damage takes the form of discontinuities of the outer envelope membrane, and results in the loss of the regular coccobacillary shape of these particles and in an increase in their size. Treatment with a combination of RNase and DNase was found to cause profound damage to all three morphological forms of the chlamydiae.