Allantoic Cavity Research Articles

The Epithelial Layer of the Term Placenta of the Asian Elephant (Elephas maximus): a Light and Scanning Electron Microscopic Study

This combined light and scanning electron microscopic (SEM) analyses are the first investigation of the fine morphology of the fetal membranes of the placenta in the Asian elephant. We used two term placentas with gestation periods of 634 days (Zoological Garden Leipzig) and 657 days (Zoo Hagenbeck) and selected unequal sites as specimens, particularly ‐ free membrane of the allantois, ‐ lateral edge of the placenta band, the ‘haemophagous zone’, ‐ allantochorion near the placental band, and allantochorion on the end of the chorioallantoic sac. Microscopically the free membrane of the allantois shows a simple, cuboidal epithelium with apical domains of microplicae and microvilli (fig. 1, inset). In the SEM analyses we documented a cobblestone‐like architecture of the epithelial cells with various cell sizes, small, middle and giant (fig. 1.). Furthermore, we found pear‐shaped cells with long pedicle, attached to the basement membrane and cell openings, presumably due to cell desquamation (fig. 2.). The haemophagous zone was characterized with a lamellate system between the long finger‐like chorionic villi and the maternal blood lacunae. The simple, squamous and columnar trophoblast cells are here bathed in the maternal blood. In some chorionic villi brown‐yellow material is deposited in the fetal connective tissue. A special result was the presence of strip‐like microplicae in the middle of chorionic villi. The allantochorion near the placental band sometimes indicated simple and ramified chorionic villi with smooth, gyrus‐like trophoblast cells and foamy cytoplasm. The attached allantois possesses a simple, columnar epithelium with microvilli. Furthermore, we observed villi‐like projections rising up to the allantoic cavity. The white ‘pustules’ on the outer surface of the end of the chorioallantoic sac consist of stratified squamous epithelia. Numerous mitoses were documentedin the basal domain of the pustules. A surprisingly result was the presence of intra‐epithelial capillaries. They invade the trophoblast cells to presume a nutrient exchange also outside of the placental band with its important functional metabolism. This study revealed unknown and interesting features of the epithelial layer of different locations in the placenta of the Asian elephant. Perhaps, with a detailed knowledge of the morphology of the epithelium we could convey a better understanding of functional cohesions of elephant placentation. image [ The free membranes of the allantois of the term placenta of the Asian elephant (Elephas maximus) shows a cobblestone‐like architecture of the simple cuboidal epithelial cells with various sizes. The surface morphology was determined through microplicae and microvilli. ] image [ Numerous pear‐shaped cells with long pedicle exist among the cuboidal epithelial cells, attached on the basement membrane. The cells possessed modifications of their surface, particularly microplicae. Some pear‐shaped cells shows only the pedicle, perhaps that is a loss through preparation or its presumable due to cell desquamation. One erythrocyte is localized between these cells. ]

The IκB Kinase Is a Key Factor in Triggering Influenza A Virus-induced Inflammatory Cytokine Production in Airway Epithelial Cells

Influenza A viruses continue to represent a severe threat worldwide, causing large epidemics and pandemics responsible for thousands of deaths every year. Excessive inflammation due to overabundant production of proinflammatory cytokines by airway epithelial cells is considered an important factor in disease pathogenesis. Here we report that influenza A virus induced IkappaB kinase (IKK) activity in human airway epithelial A549 cells, resulting in persistent activation of nuclear factor-kappaB (NF-kappaB), a critical regulator of the inflammatory response. Although lung epithelial cells are highly sensitive to stimulation of the IKK/NF-kappaB pathway by influenza virus infection, NF-kappaB was not activated in several non-pulmonary cells permissive to the virus, indicating a cell-specific response. Moreover, NF-kappaB was not essential for virus replication but triggered the expression of proinflammatory cytokines in infected lung cells and was directly responsible for production of high levels of interleukin-8, a chemokine associated with influenza-induced inflammation and airway pathology. We also report that 9-deoxy-delta9,delta12-13,14-dihydro-prostaglandin D2, a cyclopentenone prostanoid with therapeutic efficacy against influenza in preclinical studies, was a powerful inhibitor of influenza virus-induced IKK activity and interleukin-8 production by human pulmonary cells. The results identify IKK as an important factor in triggering influenza virus-induced inflammatory reactions in pulmonary epithelium, suggesting novel therapeutic approaches in the treatment of influenza.

Mycoplasma meleagridis-Induced Lesions in the Tarsometatarsal Joints of Turkey Embryos

Mycoplasma meleagridis (MM) has the ability to cause bone deformity in turkey poults. However, few pathological lesions have been described and no evidence of MM-induced damage to the bones has been shown. In this study, 17-day-old turkey embryos were inoculated with MM into the allantoic cavity. On the 27th day, eight of the 22 embryos presented with curved toes. Scanning electron microscopy of the tarsometatarsal joints showed fissures in the cartilage. Histological sections of the joints revealed only the infiltration of cells with eosinophilic granules. Immunohistochemical staining (IHS) showed the presence of MM in the aggregates of the bone marrow cells and the cells with eosinophilic granules. Some of these cells were harvested by laser capture microdissection (LCM), lysed, and used as template DNA. With a pair of MM-specific primers in a conventional polymerase chain reaction (PCR), a gene product was amplified, and it comigrated with the MM DNA, which indicates that these captured cells contained MM DNA. Thus, this research shows that inoculation of MM into the turkey embryos produced joint lesions and caused cellular infiltration within the bones.

Application and Evaluation of RT-PCR—ELISA for the Nucleoprotein and RT-PCR for Detection of Low-Pathogenic H5 and H7 Subtypes of Avian Influenza Virus

Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples originating from chickens experimentally infected with 2 different low pathogenic avian influenza viruses, from domestic ducks and from wild aquatic birds were examined. The outcome of 1) the universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA) procedure directed against NP (NP RT-PCR-ELISA) and 2) the subtype specific RT-PCR for H5 and H7 were compared to the results obtained by inoculation of the same specimens into the allantoic cavity of embryonated specific pathogen free (SPF) hen's eggs. Using inoculation in SPF fowl eggs as standard the sensitivity of the NP RT-PCR-ELISA and the RT-PCR for H5 or H7 was 91% and 94%, and the corresponding specificity 98% and 96%. In comparison with inoculation into eggs an additional of 9 samples were positive by NP RT-PCR-ELISA and 13 samples were positive by RT-PCR for one of the HA subtypes. Hence, the 3 RT-PCR procedures described are fast, sensitive and specific for detecting AIV and subtyping H5 and H7 and they are obvious alternatives when testing large numbers of samples.

Effect of grazing system on fetal development in Nellore cattle

Intensive grazing systems for beef females, based on abundant availability of high quality forages and supplementary concentrates, may affect fetal development. The objective of this study was to determine the effect of grazing system on length of gestation, fetal development, and characteristics of the calf at birth. Twenty-four pregnant (bred to Nellore bulls) Nellore females were allocated into two groups. The control group (G1) grazed Brachiaria decumbens ( signal grass) in a traditional (extensive) grazing system and the second group (G2) were managed on Panicum maximumcv. Tanzania 1 ( Tanzania grass) in an intensive grazing system. Fetal development was evaluated by ultrasonography on days 31, 45, 59, 94, 122, 220, and 255 of gestation. The diameter of the amniotic and allantoic cavities, crown-rump length, circumference, and diameter of the head and ocular orbit were determined. At birth, calves were weighed and height, length, thoracic circumference, and ocular orbit and bi-parietal diameters were measured. There were no differences ( P>0.05) in fetal development. The G1 cows had a longer gestation period (4.5 days; P<0.05) and their calves had greater ( P<0.05) weight, height, length, and thoracic circumference at birth. In conclusion, Nellore females raised under intensive pasture management conditions, had significantly shorter gestation and smaller calves at birth than those raised under extensive pasture management conditions. Therefore, adoption of new management practices (e.g. intensive pasture management), should take into consideration animal behavior and productivity.

Do pregnant lizards resorb or abort inviable eggs and embryos? Morphological evidence from an Australian skink, Pseudemoia pagenstecheri.

Although pregnant viviparous squamates are sometimes claimed to be able to resorb inviable eggs and embryos from the uterus, definitive evidence for such resorption is not available. After placing pregnant female Pseudemoia pagenstecheri into conditions under which embryonic development is terminated, we periodically harvested the gravid oviducts and examined them histologically. Females contained abnormal and degenerating eggs and embryos that had died in various stages of development. Dead embryos had undergone extensive cytolysis, dissolution, and aseptic necrosis and vitelline masses showed signs of deterioration and passage down the oviduct. The uterine mucosa lay in direct contact with the vitelline material, with no intact shell membrane intervening between them. Yolk was sometimes displaced into the exocoelom and allantoic cavity due to rupture of the extraembryonic membranes. Histological examination revealed no evidence of the uptake of yolk by the uterine epithelium or its accumulation in the subepithelial connective tissue. In many specimens, the uterine epithelium showed minuscule, apical granules. The position, appearance, and staining properties of the granules suggests them to be secretory, a manifestation of placentotrophy. Our observations indicate that P. pagenstecheri females retain dead eggs and embryos for several weeks or longer, yet do not resorb them during that period. This lizard is the second placentotrophic skink species in which resorption has been suspected, but in which abortive eggs appear to be retained or extruded instead of being resorbed by the oviducts. Researchers should not assume that squamates can digest and resorb oviductal eggs without definitive morphological evidence.

Eosinophil Induction in Chicken Embryos and its Responsiveness to the Chemoattractant fMLP

Inoculation of concanavalin-A (Con-A) into the allantoic cavity of chicken embryos resulted in a massive infiltration of eosinophilic granulocytes into the spleens. Immunohistochemical staining of formalin-fixed, paraffin-embedded spleen sections with monoclonal anti-major basic protein (MBP) serum revealed that these cells contain MBP, and were therefore eosinophils. Transmission electron microscopic examination of the spleens showed that these cells contained bi-lobulated nuclei and many granules of various sizes and shapes. These granules, however, did not contain a central core, which is a prominent feature in their mammalian counterparts. The eosinophils isolated from adult chickens underwent remarkable surface changes when incubated with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) by forming and extending surface projections. Adult eosinophils were immotile, but showed chemotactic response when fMLP was used as a chemoattractant in chemotaxis assays.

Clinical epidemiologic and experimental evidence for the transmission of Newcastle disease virus through eggs.

Sporadic outbreaks of Newcastle disease (ND) occurred in Taiwan during 1998-2000. In some cases, the disease occurred in broilers less than 2 wk old that originated in a broiler breeder farm, so spread of the ND virus (NDV) from the infected breeder farm to broiler ranches was suspected. The purpose of the present study was to examine the possibility of the transmission of NDV through eggs. Both clinical and experimental evidence were used to prove that this is possible. From epidemiological investigation, the possibility of transmission through eggs was suggested in two separate ND cases from a breeder farm and its progeny because two identical NDVs were isolated from both cases. In order to clarify the possibility of the transmission through eggs, one mean egg lethal dose (ELD50) of NDV was inoculated into the allantoic cavity of 155 9-to-11-day-old specific-pathogen-free (SPF) chicken embryos. Seventy-one hatching chicks from the inoculated embryos were raised for 14 days. The cloacal swabs from those chicks at the ages of 1, 4, and 7 days and the tissues after necropsy at the ages of 14 days were taken for virus isolation. The same NDV was reisolated from three hatching chicks. This experiment confirms that a few chicken embryos infected in ovo with a low titer of NDV can hatch and contain NDV after hatching, which results in NDV spreading through eggs.

Thermostabilization of egg grown influenza viruses

The observation that the addition of deuterium oxide to tissue culture cell harvests stabilized trivalent oral poliovirus vaccine prompted us to examine the effect of deuterium oxide on stabilization of other licensed and experimental live viral vaccines. The most striking effect afforded by deuterium oxide was on stabilization of live, attenuated influenza A and B vaccine candidates grown in the allantoic cavity of embryonated eggs. Thermostabilization with deuterium oxide is much greater than seen with standard stabilization of these live influenza vaccines with sucrose–phosphate–glutamate. Subsequently, we have shown that a similar, stabilization is provided by diluting egg allantoic fluid in water or minimal essential medium. The mechanism of action has been explored but remains uncertain. The implications of being able to stabilize influenza after harvesting from eggs have practical application for utilization of live influenza vaccines and provides a possible way to increase and standardize the potency of inactivated vaccines that may partially degrade in the inimical environment of allantoic fluid during growth and before final vaccine purification and stabilization.

Intestinal damage in gastroschisis correlates with the concentration of intraamniotic meconium

Background/Purpose: Contact with amniotic fluid (AF) causes intestinal damage in gastroschisis, which has been shown to be caused by intraamniotic meconium. However, whether this intraamniotic meconium–induced intestinal damage is concentration dependent has not been investigated previously. The purpose of this study is to investigate the effects of intraamniotic human meconium at various concentrations on the intestines of chick embryo with gastroschisis. Methods: Five-day-old fertilized chick eggs were used. Gastroschisis was created through the amniotic cavity without opening the allantoic cavity. Sterile meconium was obtained from newborn humans. Meconium suspensions at various concentrations were prepared using saline and instilled into the amniotic cavity. Results: Intraamniotic 1:200 and 1:400 meconium was found to cause intestinal damage. Meconium concentrations lower than 1:400 did not cause intestinal damage. Histopathologic examination of the intestines of the 1:200 and 1:400 meconium groups showed serosal thickening, inflammation, focal fibrin, and collagen deposits. Histopathologic features of the intestines of the 1:600 and 1:800 meconium groups did not differ from the intestines of the control group. Conclusion: Intraamniotic meconium, which is responsible for intestinal damage in gastroschisis, must reach a threshold level to induce intestinal damage. J Pediatr Surg 36:1811-1815. Copyright © 2001 by W.B. Saunders Company.

Primary target cells of virulent strains of type A influenza virus in chicken embryos

Virulent or avirulent strains of type A influenza virus were inoculated into the allantoic cavities of chicken embryos. The antigens of virulent strains appeared initially in the surface epithelium of the allantoic membrane, then in vascular endothelial cells of the chorioallantoic membrane and visceral organs of the embryos, and then spread to parenchymal cells of many organs. In contrast, the antigens of the avirulent strain were confined to the allantoic membrane. These observations indicate that the primary target of virulent influenza viruses in chicken embryos is vascular endothelial cells, and that the embryos died after systemic viral infection.

Recovery of a Virulent Strain of Newcastle Disease Virus from Cloned cDNA: Expression of a Foreign Gene Results in Growth Retardation and Attenuation

A recombinant mesogenic NDV strain, Beaudette C, and an engineered recombinant NDV expressing an additional gene were generated entirely from cloned cDNAs. For this purpose, a full-length cDNA clone of the virus genome, represented in eight different subgenomic fragments, was assembled in a transcription plasmid between a T7 RNA polymerase promoter and a hepatitis delta virus ribozyme sequence. Infectious NDV could be generated in the cells infected with recombinant vaccinia virus, which expressed T7 RNA polymerase, by simultaneous expression of antigenome-sense NDV RNA from the full-length plasmid and NDV NP, P, and L proteins from cotransfected plasmids. Recombinant virus was then amplified and recovered, either after inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free eggs or after further passage in cell culture. Characterization of the recombinant NDV showed similarities in growth and pathogenicity to that of the parental wild-type virus. By using this system, a recombinant NDV containing a foreign gene encoding chloramphenicol acetyltransferase (CAT) was generated. To do this, the CAT transcription cassette containing the CAT open reading frame, flanked by NDV gene start and gene end sequence motifs, was inserted into the region between the HN and L genes of the full-length cDNA. This construct was then used in the generation of a recombinant NDV expressing CAT protein. The CAT gene was maintained stably for at least eight passages without any detectable loss of the gene from the recombinant. Generation of the recombinant virus, however, was associated with reduced plaque size, slower replication kinetics, and more than 100-fold decrease in yield. In addition, the virus showed an increase in mean death time for eggs and a lower intracerebral pathogenicity index in day-old chicks, implicating attenuation of the recombinant virus. Thus, introduction of an additional gene into the NDV genome represents a method to achieve growth retardation and attenuation. These results also indicate that NDV can be engineered to express foreign protein stably and can be manipulated in the future for use as a vaccine vector.

Chicken Embryo Lethality Assay for Determining the Virulence of Avian Escherichia coli Isolates

Multiple isolates of Escherichia coli from clinical cases of colibacillosis and E. coli from the intestinal tracts of normal broilers at slaughter were assayed by the embryo lethality test to determine their virulence. The assay was repeated five times in order to establish reproducibility and determine the statistical parameters of the test. This study showed that the inoculation of approximately 100 colony-forming units in the allantoic cavity of 12-day-old embryos discriminated between virulent and avirulent E. coli isolates. Gross lesions included cranial and skin hemorrhages in addition to encephalomalacia in embryos inoculated with virulent isolates. Abnormalities were observed by microscopic examination of the heart, brain, and liver in embryos inoculated with virulent isolates. Analysis of data indicated that the length of the test should be 4 days. In the virulent group, day 2 postinoculation had the most significant death patterns. Sample size calculations indicated that 11 embryos are sufficient for the assay. On the basis of death rates, isolates considered to be avirulent had an embryo death rate of <10%, moderately or secondary pathogens had a 10%-29% death rate, and virulent isolates had a death rate of >29%. An important aspect of this assay is the accessibility of good-quality fertile embryonated eggs.

Effect of esophageal ligation on amniotic fluid volume and urinary flow rate in fetal sheep

Objective:Although the fetus normally swallows large volumes of amniotic fluid each day, it is unclear whether amniotic fluid volume increases after fetal esophageal obstruction or whether fetal urine production changes. Our objective was to determine the effects of fetal esophageal ligation on amniotic fluid volume and urinary flow rate over time. Study Design:Seven late-gestation fetal sheep underwent esophageal ligation, and 7 served as time Controt animals. The urachus was ligated to eliminate urine flow to the allantoic cavity. On days 1, 3, 5, 7, and 9 after surgery, we measured the composition of amniotic fluid, fetal urine, and fetal and maternal blood, as well as amniotic fluid volume and fetal urinary flow rate. A 3-factor analysis of variance was used for statistical analysis. Results:Amniotic fluid volume did not change with time in the control group, averaging 876 ± 142 mL (meant SEM), and it decreased in the esophageal ligation group (P =.020), averaging 309 ± 75 mL on day 9. Fetal urinary flow rate was lower (P = .0063) in the esophageal ligation group (431 ± 27 mL/d) than in the control group (631 ± 54 mL/d). There were no differences in fetal or maternal blood compositions between the two groups. Amniotic fluid sodium and chloride increased in the ligated animals. Conclusions:Polyhydramnios did not occur after esophageal ligation, even though the fetuses excreted approximately 4000 mL of urine over the 9-day study period. This suggests that intramembranous absorption is substantially increased. With only small changes in amniotic solute concentrations, intramembranous solute absorption must occur simultaneously with water, suggesting a near-zero reflection coefficient for solutes. We speculate that fetal urine, lung secretions, or both contain a factor that increases intramembranous permeability.

The effects of intraamniotic human neonatal urine and meconium on the intestines of the chick embryo with gastroschisis

Background/Purpose: Urinary waste products in the amniotic fluid has been implicated as a cause of intestinal damage (ID) in gastroschisis based on the fact that fetus urinates physiologically into the amniotic cavity. However, experimental and clinical data suggest that intrauterine defecation is a physiological event, thus gastrointestinal waste products also may be responsible for ID in gastroschisis. An experimental study was performed to investigate the effects of intraamniotic human neonatal urine and diluted meconium on the intestines of chick embryo with gastroschisis.Methods: Five-day-old fertilized chick eggs (Gallus domesticus) were used. Gastroschisis was created through amniotic cavity without opening the allantoic cavity. Sterile urine and meconium were obtained from newborn humans, and 1% meconium suspension was prepared. The eggs were divided in to 3 groups. In the first group, gastroschisis was created, and amniotic fluid was reinstilled without changing its composition (control group). Equal amounts of amniotic fluid and urine mixture was instilled into the amniotic cavity in second group (urine group) and 1% meconium suspension was instilled in similar fashion in the third group after creation of gastroschisis (meconium group).Results: Histopathologic features of the intestines of the urine group did not differ from the intestines of the control group. The meconium group's bowel showed serosal thickening, inflammation, focal fibrin, and collagen deposits. Histopathologic changes of intestines induced by intraamniotic diluted meconium are consistent with the ones described for human gastroschisis specimens.Conclusion: Gastrointestinal waste products seem responsible for the ID in gastroschisis rather than urinary waste products.

Generation of recombinant lentogenic Newcastle disease virus from cDNA

Recombinant lentogenic Newcastle disease virus (NDV) of the vaccine strain Clone-30 was reproducibly generated after simultaneous expression of antigenome-sense NDV RNA and NDV nucleoprotein, phosphoprotein and RNA-dependent RNA polymerase from plasmids transfected into cells stably expressing T7 RNA polymerase. For this purpose, the genome of Clone-30, comprising 15186 nt, was cloned and sequenced prior to assembly into a full-length cDNA clone under control of a T7 RNA polymerase promoter. Recombinant virus was amplified by inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free (SPF) chicken eggs. Two marker restriction sites comprising a total of five nucleotide changes artificially introduced into noncoding regions were present in the progeny virus. The recombinant NDV was indistinguishable from the parental wild-type virus with respect to its growth characteristics in cell culture and in embryonated eggs. Moreover, an intracerebral pathogenicity index of 0.29 was obtained for both viruses as determined by intracerebral inoculation of day-old SPF chickens, proving that the recombinant NDV is a faithful copy of the parental vaccine strain of NDV.

Selection of Receptor-Binding Variants of Human Influenza A and B Viruses in Baby Hamster Kidney Cells

Cultivation of human influenza viruses in the allantoic cavity of embryonated chicken eggs leads to a selection of receptor-binding variants with amino acid substitutions on the globular head of the hemagglutinin (HA) molecule. Such selection can be avoided by growing the human viruses in Madin Darby canine kidney (MDCK) cells. In the present study, we tested whether baby hamster kidney (BHK) cells select receptor-binding mutants of human influenza viruses. After isolating H1N1, H3N2, and type B influenza viruses from clinical samples in MDCK cells, we passaged them in either BHK cells or chicken eggs. The BHK-grown viruses differed from their MDCK-grown counterparts by virtue of mutations in the HA: 225D → G (H1N1 virus), 128T → A and 226I → V (H3N2), and 187N → D (type B) (H3 numbering). Variants with different substitutions were selected by passaging of the same MDCK-grown parents in eggs: 141L → H, 208R → H, and 225D → G (H1N1), 194L → I (H3N2), and 137G → R (B). Compared with their MDCK-grown counterparts, both BHK- and egg-grown viruses possessed a higher affinity for the cellular membranes of BHK cells and of the chorioallantoic cells of chicken embryos and for a 3′-sialylgalactose-containing synthetic sialylglycopolymer. By contrast, changes in the affinity of mutants for a 6′-sialyl-(N-acetyllactosamine)-containing sialylglycopolymer varied from negative to positive. Fluorescence-activated cell-sorting analysis with linkage-specific lectins showed that the density of the 6′-sialyl-(N-acetyllactosamine)-containing receptors is substantially lower on the surface of BHK cells than on MDCK cells, providing an explanation for the growth restriction of human viruses in the former cells. Our data demonstrate that cultures of BHK cells, like eggs, can select receptor-binding variants of human influenza viruses.

Allantoic Fluid Compositional Changes During Acute Urine Drainage in Fetal Sheep

This study examines the effects of ovine fetal urine drainage, which prevents urine inflow into the allantoic and amniotic cavities, on allantoic and amniotic fluid osmolalities and electrolyte concentrations in an attempt to study solute movement out of the allantoic cavity. Nine chronically catheterized fetal sheep at 132 +/- 2 (mean +/- standard error) [SE] days' gestation, with allantoic and amniotic cavity catheters, were studied. Seven animals were studied for 9 hours, with maternal and fetal blood drawn hourly, and amniotic and allantoic fluid sampled for osmolality and electrolyte concentrations. On the second day, seven animals (five from the above group) were studied in the same fashion, except that fetal urine was drained after the first hour. Allantoic and amniotic fluid osmolality increased significantly (13.2 +/- 1.1 (SE) mOsm/kg, P < .0001, 7.5 +/- 1.5, P < .03, respectively) after fetal urine drainage but not sufficiently enough to account for the large amount of fetal urine diverted (217 mL per 8 hours). Fetal (3.1 +/- 0.6, P < .04) and maternal plasma (2.4 +/- 0.5, P < .03) osmolalities increased significantly, and this increase was consistent with the amount of fetal urine drained over the 8-hour drainage period. Allantoic (4.0 +/- 1.7 mEq/L, P < .003) and amniotic sodium (4.2 +/- 1.5 mEq/L, P < .03) and amniotic chloride (3.1 +/- 1.6 mEq/L, P < .04) increased significantly as compared with control animals. During the fetal urine drainage period, the increases in allantoic and amniotic fluid osmolalities and electrolyte concentrations confirmed that water left these compartments via the intramembranous pathway. If only water (and not solutes) had left these compartments, then the increase in osmolalities would have been too small to account for the large volume of diverted fetal urine. Therefore, solutes, in addition to water, must leave the allantoic cavity through the intramembranous pathway as no other pathway exists. Furthermore, solute movement through the intramembranous pathway may explain the ability of the fetus to maintain molecular and solute gradients between fluid compartments. Finally, the intramembranous pathway may play an important role in amniotic and allantoic fluid volume regulation and composition.

Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.

Human influenza viruses are more efficiently isolated by inoculating patient samples into the amniotic rather than the allantoic cavity of embryonated chicken eggs. This type of cultivation selects virus variants with mutations around the hemagglutinin (HA) receptor binding site. To understand the molecular basis of these phenomena, we investigated the abundances of sialic acid (SA) linked to galactose (Gal) by the alpha-2,3 linkage (SA alpha2,3Gal) and SA alpha2,6Gal in egg amniotic and allantoic cells and in Madin-Darby canine kidney (MDCK) cells. Using SA-Gal linkage-specific lectins (Maackia amurensis agglutinin specific for SA alpha2,6Gal and Sambucus nigra agglutinin specific for SA alpha2,3Gal), we found SA alpha2,3Gal in both allantoic and amniotic cells and SA alpha2,6Gal in only the amniotic cells. MDCK cells contained both linkages. To investigate how this difference in abundances of SA alpha2,3Gal and SA alpha2,6Gal in allantoic and amniotic cells affects the appearance of host cell variants in eggs, we determined the receptor specificities and HA amino acid sequences of two different patient viruses which were isolated and passaged in the amnion or in the allantois and which were compared with MDCK cell-grown viruses. We found that the viruses maintained high SA alpha2,6Gal specificities when grown in MDCK cells or following up to two amniotic passages; however, further passages in either the amnion or allantois resulted in the acquisition of, or a complete shift to, SA alpha2,3Gal specificity, depending on the virus strain. This change in receptor specificity was accompanied by the appearance of variants in the population with Leu-to-Gln mutations at position 226 in their HA. These findings suggest that lack of SA alpha2,6Gal linkages in the allantois of chicken eggs is a selective pressure for the appearance of host cell variants with altered receptor specificities and amino acid changes at position 226.

Mast Cell Activation Accelerates the Normal Rate of Angiogenesis in the Chick Chorioallantoic Membrane

Using shell-less cultures of chick embryos, the effect of mast cell degranulation on normal rates of angiogenesis in the chorioallantoic membrane (CAM) was recorded from Day 6 to Day 9. Based on the centripetal ordering method with capillaries as the initial point of reference, the first two orders of pre- and postcapillary microvessels were evaluated. After infusion of compound 48/80 into the allantoic cavity, the normal proliferation rate of order-1 pre- and postcapillaries was markedly increased. Increases of second-order vessel numerical densities (number/cm2) were less than those of the order-1 vessels. Thus, length densities (mean length × numerical density) were increased for the order-1 vessels only. During the accelerated rate of CAM angiogenesis, endothelial restriction of FITC–dextran 150 remained uniformly high. Hence, secreted mast cell products likely worked synergistically with endogenous CAM angiogenic factors between Days 6 and 9, without increasing rates of macromolecular extravasation. However, histamine injection into the allantoic cavity at Day 10, in conjunction with calcium supplementation of the shell-less cultures at Day 9, elevated the rate of FITC–dextran 150 extravasation. Whether differentiation of this transendothelial pathway is required for enhancement of CAM angiogenesis after Day 10 remains uncertain.