Chemotaxis in Mycoplasma gallisepticum.

Boyden-type chemotactic chambers were used to demonstrate that Mycoplasma gallisepticum (MG) was capable of migrating into chemotactic membranes. Scanning electron microscopy was used to confirm that MG could penetrate the membranes. To further demonstrate the invasive ability of MG, MG was deposited on the shell membranes of 9-day-old chicken embryos, and after 6 days of incubation, the presence of MG DNA in the allantoic fluids was detected by polymerase chain reactions. These results indicate that MG can penetrate cellular membrane, possibly by going through the porous cellular surface.

In vivo internalization of Staphylococcus aureus by embryonic chick osteoblasts.

Staphlylococcus aureus is the primary pathogen associated with osteomyelitis, an acute and recurrent bone disease. Internalization of S. aureus by cultured embryonic chick calvarial osteoblasts has been observed. The purpose of this study was to demonstrate that internalization of bacteria by embryonic chick calvarial and tibial osteoblasts occurs in vivo. In initial experiments, 10(8) colony forming units (cfu) of S. aureus, strain UAMS-1 or Cowan 1, were injected subcutaneously under the scalp skin of 17 day chick embryos. After 45 min, calvariae were harvested and processed for transmission electron microscopy (TEM). In subsequent experiments, 10(9) cfu of UAMS-1 were injected into the allantoic sac of 17 day chick embryos via a small opening in the egg shell. After 48 h, calvariae and tibiae were harvested for TEM. S. aureus cells were found in approximately 14% of the calvarial osteoblasts after subcutaneous injection and in 11% of calvarial and tibial osteoblasts following intraallantoic injection. Endosomes were observed in some cells, but most bacteria internalized appeared to be free in the cytoplasm. Osteoblasts with as few as five bacteria had a greater loss of cytoplasmic integrity and a more heterochromatic nucleus than osteoblasts with fewer bacteria or than uninfected osteoblasts. S. aureus cells in calvariae and tibiae were also observed in the cytoplasm of approximately 4% of the osteocytes in mineralized bone matrix. Thus, internalization of S. aureus by osteoblasts in vivo augments the previous observation in vitro. This study has also shown that osteoblasts with few bacteria continue differentiating into osteocytes. Results of these experiments support the hypothesis that internalization of S. aureus by osteoblasts may play a role in the etiology of osteomyelitis.

Changes in the virulence of Mycobacterium avium after passage through embryonated hens' eggs.

Eight-day-old embryonated hen's eggs were used as a model to study Mycobacterium avium virulence. Strains isolated from human patients caused 20-90% mortality when eggs were infected by injection of bacterial suspensions into the amniotic sac. Virulence of examined strains subsequently decreased with passage through eggs to between 0 and 40% mortality in four passages. Virulence of the egg-attenuated strains could be restored by passage through human peripheral blood mononuclear cells. The site of infection in the egg was usually the mesodermal layer of the chorioallantoic membrane. A few small granulomas containing acid-fast bacteria were seen in the liver, but not in other organs. Death of chicken embryos may have resulted from destruction of the mesodermal layer of the chorioallantoic membrane with consequent respiratory failure. PBMCs infected with less virulent egg-passaged strains of M. avium produced higher levels of tumor necrosis factor-alpha than did peripheral blood mononuclear cells infected with more virulent nonpassaged strains.

Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis.

The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis. Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD50 of 10(3) colony forming units (CFU) or greater (low virulence) from those having an LD50 of approximately 10(1) or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica, a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD50 of 10(2) were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive.

Effect of host lineage on the virulence of Campylobacter jejuni/coli in the chick embryo model.

The chorioallantoic membrane (CAM) inoculated chick embryo model was used to study the effect of host lineage on the virulence of Campylobacter jejuni and Campylobacter coli. LD50 values were used to compare the susceptibilities of chick embryos from eight inbred chicken lines to infection by four strains of C. jejuni and one strain of C. coli. Differences in susceptibility were found between inbred chicken lines. These were shown not to be due to maternal antibody status, nor transfer of antibody to the developing embryo. Susceptibility to infection was also found to vary according to the Campylobacter strain used. These results indicate that both the bacterial strain and host lineage of the chicken line used affect resistance to infection in the CAM inoculated chick embryo model.

Use of temperature-sensitive gel for concentration of influenza virus from infected allantoic fluids.

Cross-linked, poly(N-isopropylacrylamide) gel was used to concentrate avian influenza virus from allantoic fluid. Placing the gel in virus-infected allantoic fluid at 4 degrees C caused the gel to swell and absorb small molecular weight solutes, while excluding avian influenza virus and other large particles. Warming the gel to 37 degrees C or more caused the gel to collapse. The gel remained functional after sterilization in an autoclave and could be reused to concentrate other samples of allantoic fluid. Using a combined concentration and elution technique, we were able to achieve an average of 84.2% virus recovery, while reducing the fluid volume from 90 ml to 7.6 ml.

Investigations on the pathogenicity of Listeria spp. by experimental infection of the chick embryo.

To investigate the pathogenicity of Listeria spp., chick embryos were infected by two methods. Very encouraging results were obtained with the inoculation of the chorioallantoic membrane (CAM) of 10 day old chick embryos: embryos inoculated with pathogenic strains (L. monocytogenes or L. ivanovii) died within 72 h, whereas those infected with apathogenic strains survived. This test could be a suitable method to replace the mouse pathogenicity test.

Genetic susceptibility of indigenous chicks to subgroup A Rous sarcoma virus inoculated via the chorioallantoic membrane.

An investigation was made using chicks of two Indian indigenous breeds of fowl, Kadaknath and Aseel, to ascertain genetic resistance to infection by Rous sarcoma virus of subgroup A. A standard inoculation dose of 0.2 ml virus containing 1000 pock forming units ml-1 was injected via the chorioallantoic membrane (CAM) into the 11-day-old embryos that were subsequently hatched. The sensitivity of the two indigenous breeds was compared with the highly susceptible exotic White Leghorn (WL) strain maintained in the laboratory. The Kadaknath breed was about three-fold and Assel, about six-fold less sensitive than the WL strain, indicating superiority of the indigenous breeds over the exotic breed of fowl. Most of the CAM-susceptible chicks died of liver tumour (LT) and most of the CAM-resistant chicks survived. However, conversely associated tumour phenotype subclass chicks, i.e. CAM-susceptible LT-negative chicks that survived and CAM-resistant LT-positive chicks that died, occurred consistently in the three breeds of fowl. Nevertheless, the overall survival potential of Kadaknath chicks measured up to 8 weeks post-hatching was greater than that of Aseel chicks. Neither transformation of embryonic tissue prior to hatching nor the visceral metastasis including liver conformed with the degree of CAM-infection as measured by number of pocks on CAMs.

Comparison of in vitro replication and cytopathology caused by strains of canine distemper virus of vaccine and field origin.

Three biological properties of canine distemper virus were examined to determine if any would consistently differentiate field from vaccine strains of the virus. The properties were the ability to (1) infect macrophages and epithelial cells, (2) produce distinct cytopathologic effect in alveolar and peritoneal macrophages and Vero cells, and (3) produce pocks on the chorioallantoic membrane of embryonated chicken eggs. Four vaccine strains and 5 field isolates were used in the study. The 5 field isolates were obtained directly from canine tissues. Of the 3 properties studied, only the comparison of the ability of the viruses to infect macrophages and epithelial cells was a consistent marker of virus origin. Virulent field isolates would only infect macrophage cultures, whereas the vaccine strains infected both types of cells. One avirulent field isolate from a case of old dog encephalitis reacted more like a vaccine strain by infecting both cell types.

Etiology and pathology of equine placentitis.

Placentas from aborted, stillborn, and premature foals were examined during the 1988 and 1989 foaling seasons, and 236 of 954 (24.7%) had placentitis. Microorganisms associated with placentitis were isolated or demonstrated from 162 of 236 (68.6%) placentitis cases. Leptospira spp. and a nocardioform actinomycete were 2 important, newly emerging bacteria associated with equine placentitis. Major pathogens identified in decreasing order were Streptococcus zooepidemicus, Leptospira spp., Escherichia coli, a nocardioform actinomycete, fungi, Pseudomonas aeruginosa, Streptococcus equisimilis, Enterobacter agglomerans, Klebsiella pneumoniae, and alpha-hemolytic Streptococcus. Pathogens were not recovered in 64 cases (27.1%) and overgrowth by saprophytic bacteria was recorded in 10 cases (4.2%). Twenty-seven cases (16.6%) had mixed bacterial growth and 93 cases (57.4%) had bacteria cultured from both placenta and fetal organs. The majority of the placentitis cases caused by bacteria, with the exception of Leptospira spp. and the nocardioform actinomycete, occurred in 2 forms. One was acute, focal or diffuse; had an infiltration of neutrophils in the intervillous spaces or necrosis of chorionic villi; was associated with bacteremia; and frequently occurred in the placenta from fetuses expelled before or at midgestation. The other was observed from foals expelled at late gestation, was mostly chronic and focal or focally extensive, and occurred mostly at the cervical star area. Chronic placentitis was characterized by the presence of 1 or a combination of the following lesions: necrosis of chorionic villi, presence of eosinophilic amorphous material on the chorion, and infiltration of mononuclear inflammatory cells in the intervillous spaces, villous stroma, chorionic stroma, vascular layer, and allantois.(ABSTRACT TRUNCATED AT 250 WORDS)

Salmonella typhimurium penetration through the eggshell of hatching eggs.

This study was undertaken to demonstrate penetration of Salmonella typhimurium through the eggshell of newly laid broiler hatching eggs. Eggs were challenged either by lightly spraying the bacteria over the blunt end of the egg or by contact with contaminated dry nest litter. Exposure time for both groups was 10 minutes; afterward, all eggs were disinfected and incubated 19 days under normal conditions. Chorioallantoic membranes and yolk sacs were cultured in brain-heart infusion broth on day 19 to demonstrate penetration. Isolation of the bacteria from chorioallantoic membranes and yolk sacs, respectively, were as follows: sprayed group: 100% and 83%; contact group: 59% and 29%. These results showed that although water enhanced S. typhimurium penetration, its presence on the eggshell is not essential for penetration to occur.

Chicken embryo propagation of type I avian adenoviruses.

Forty-two clone-purified, cell-culture-propagated type I avian adenoviruses (AAV) representing 11 serotypes and two intermediate strains were evaluated for virus replication (evidenced by embryo death and lesions) resulting from the inoculation of specific-pathogen-free chicken embryos via the chorioallantoic sac or yolk sac. Commonly observed embryonic changes were death, stunting and curling, hepatitis, splenomegaly, congestion and hemorrhage of body parts, and urate formation in the kidneys. Basophilic or eosinophilic intranuclear inclusion bodies characteristic of fowl adenoviruses were observed in hepatocytes. The magnitude and relative uniformity of intra- and interserotypic embryo mortality, gross lesions, and virus titers was greater in embryos inoculated via the yolk sac. This work identifies the yolk sac as a practical and sensitive chicken embryo inoculation route for poultry diagnosticians to employ. It is suggested that the yolk sac may be a reliable alternative to cell culture for the successful isolation of all type I avian adenoviruses.

Infectious bronchitis virus detection in allantoic fluid using the polymerase chain reaction and a DNA probe.

A rapid extraction procedure was developed to purify infectious bronchitis virus (IBV) RNA from the allantoic fluid of inoculated embryonating eggs. Reverse transcription of viral RNA and the polymerase chain reaction (PCR) were used to amplify the viral genome from eight different strains of IBV comprising five different serotypes. A biotinylated DNA probe, prepared to a sequence within the PCR amplification product of the Beaudette strain of IBV, was used in a dot-hybridization assay; it detected the amplification products of all of the IBV strains examined. Reverse transcription and PCR amplification were judged to be specific for IBV. This was because amplification products were not detected by agarose gel electrophoresis or by dot-hybridization when template used in the PCR was extracted from allantoic fluid and the chorioallantoic membrane of uninoculated embryonating eggs or from allantoic fluid of embryonating eggs inoculated with other chicken upper respiratory viruses.

A monoclonal antibody-based immunoperoxidase procedure for rapid detection of infectious bronchitis virus in infected tissues.

A monoclonal antibody (MAb)-based indirect immunoperoxidase (IP) procedure was applied to detect infectious bronchitis virus (IBV) antigen in frozen tissue sections. With this procedure, IBV antigen could be detected in chorioallantoic membranes (CAMs) within 15 hr postinoculation and in the respiratory tissues of chickens within 96 hr postinoculation. Endogenous peroxidases in chicken tissues were removed by treatment with periodic acid. The IP technique was highly sensitive, convenient, and economical. It allowed simultaneous evaluation of antigen-bearing cells and the general tissue morphology. The IP-stained slides also provided a permanent record.

Egg dipping in hydrogen peroxide solution to eliminate Salmonella typhimurium from eggshell membranes.

The effectiveness of egg dipping in a 6% hydrogen peroxide solution to eliminate Salmonella typhimurium from eggshell membranes was evaluated. The first step was to assess the water uptake from broiler hatching eggs dipped in tap water once or twice using a positive-pressure differential method in order to determine the best method to use in the disinfection trials. Double dipping increased water uptake by 86% more than single dipping. Dipping S. typhimurium-contaminated eggs twice in a 6% hydrogen peroxide solution reduced the average number of organisms in eggshell membranes by 95% and the number of S. typhimurium-positive eggs by 55% compared with the infected untreated group. Dipping the eggs in a 6% hydrogen peroxide solution did not adversely affect hatchability.

Pasteurella multocida infection in the chicken embryo.

Pasteurella multocida infection in embryonated chicken eggs was studied by chorio-allantoic membrane inoculation. Strain differences were demonstrated in terms of lesion severity and time to death, especially during the first 24 h post-inoculation. A strain of low virulence gave a clear dose response but more virulent strains did not. Comparable results were obtained by infecting 6-week-old chickens. The main lesions in inoculated embryos appeared as severe vascular involvement of the entire embryo and feather tracts, thickening of the chorio-allantoic membrane, and enlargement and congestion of the yolk sac. The bacteria were demonstrated by transmission electron microscopy, either extracellularly or multiplying intracellularly in hepatocytes, heart tissue, and in the hyperplastic layer of the chorio-allantoic membrane, with resulting damage to the cellular organelles, and severe tissue changes.

Interaction of bovine chorioallantoic membrane explants with three strains of Brucella abortus.

Chorioallantoic membrane (CAM) explants were used to determine the in vitro growth and cytotoxic potential of 3 strains of Brucella abortus. Bovine CAM explants were inoculated with 2 x 10(7) colony-forming units of the pathogenic strain 2308, attenuated strain 19, or the rough strain RB51 of B abortus. After inoculation, the explants were harvested and examined at 2 or 4 hours, 12 or 14 hours, and 24 or 26 hours of incubation. Bacterial growth associated with each explant was determined by counting colony-forming units. The degree of cellular damage in each explant associated either with bacterial growth or bacterial toxins was evaluated by morphometric analysis after trypan blue staining. Significant differences were not detected in the numbers of bacteria of any strain of B abortus in the CAM explants at comparable time intervals. The rate of growth of the bacteria in CAM explants was higher between 2 and 12 hours after inoculation than between 12 and 24 hours after inoculation. Cytotoxic effects associated with strain 2308 were significantly (P less than 0.05) greater than that caused by other strains. Cytotoxic effects associated with strain 19 and rough strain RB51 were similar, and both were significantly (P less than 0.05) greater than the phosphate buffer solution control. Chorioallantoic membrane explants inoculated with a filtrate of heat-killed strain 2308 induced minimal cellular damage, compared with that caused by the viable bacteria. These results indicated that the number of B abortus in trophoblasts was independent of the degree of cellular damage.

Concentration and purification of Newcastle disease virus by microfiltration and exclusion liquid chromatography.

Newcastle disease virus (NDV) was concentrated and purified by microfiltration and exclusion liquid chromatography (ELC) on macroporous glass (MPG). The purified NDV eluted as one peak; its yield was strain-dependent, the degree of purification was 95% to 99%.

Inhibition of subtilisin by influenza virus infected allantoic fluids.

Allantoic fluids harvested from embryonated chicken eggs infected with reference strains of influenza A viruses were analysed for subtilisin inhibitor activity. While all acid heat-treated and nontreated virus-infected fluids could reduce subtilisin activity, fluids of FM and Bangkok strains had the greatest inhibitory ability. The degree of subtilisin inhibition closely paralleled the appearance of infectious Bangkok and FM virus in allantoic fluid. Maximum levels were achieved at 48 hr post-infection (p.i.) Ultracentrifugation analyses indicated that the bulk of thermostable inhibitor(s) of 48 hr Bangkok and FM infectious fluids remained in the supernatant rather than sedimenting with the viral pellet.

A simple and rapid characterization of influenza virus isolates by monoclonal antibodies in radioimmunoassay.

Radioimmunoassay (RIA) with infectious allantoic fluid directly bound to solid phase, suitable for detection and further characterization of influenza virus isolates, is described. This simple and rapid method was applied for description of isolates obtained from different regions of Czechoslovakia during influenza epidemic in 1983. The results confirmed that all 13 examined isolates represent influenza A viruses possessing H3 subtype haemagglutinin very similar to haemagglutinin of influenza viruses A/Bangkok/1/79 (H3N2), A/Belgium/2/81 (H3N2) and A/Philippines/2/82 (H3N2), respectively.