Pathogenesis and immunology of experimental gonococcal infection: role of iron in virulence.

Addition of iron componds to inocula of the relatively avirulent T3 or T4 colony types of gonococci increased their lethality for chicken embryos after intravenous inoculation but had little or no effect on the highly virulent T1 or T2 types. The toxicity of nonviable inocula, killed cells or sonicates, was not significantly affected by ecogenous iron. Addition of the iron-binding protein conalbumin reduced or delayed the lethal effect of T1, but not T3, gonococci although growth of both colony types in the allantoic cavity of the embryo was inhibited by this protwin. This effect can be attributed specifically to deprivation of iron since the iron-complexed form of conalbumin had no apparent influence on growth or virulence. The results indicate that the ability to acquire iron in vivo is a significant factor in gonococcal virulence. The virulent colony types appear to have enhanced ability to compete with the host for iron and this may be related to the presence of pili, other surface components, or the synthesis of iron-chelating compounds.     

Effect of siderophores on virulence of Neisseria gonorrhoeae.

The virulence of Neisseria gonorrhoeae for chicken embryos can be modified in a predictable manner by the addition of microbial siderophores to the inoculum. "Meningobactin" and "gonobactin," siderophores isolated from iron-limited cultures of meningococci and gonococci, respectively, enhance the virulence of the relatively avirulent colony type 3 (T3) organisms, but have essentially no effect on the virulence of T1 organisms. Both of these compounds were found previously to stimulate in vitro growth of the pathogenic Neisseria spp. under conditions made iron limiting by the addition of conalbumin, the transferrin counterpart of chickens. Similarly, ferrated schizokinen and arthrobactin, both dihydroxamate siderophores which stimulated growth in iron-limited conditions in vitro, also enhanced virulence of T3 organisms, whereas desferrioxamine B mesylate (Desferal), a trihydroxamate previously shown to be inhibitory in vitro, decreased the virulence of the T1 colony form. This was due to the iron-binding function of the molecule, as the iron-saturated form, ferrioxamine B mesylate, did not affect virulence. An additional trihydroxamate siderophore, ferrichrome A, which was inactive on Neisseria spp. in either the deferri- or ferrated forms in vitro, likewise did not affect virulence in the chicken embryo model. The neisserial siderophores were more effective than the other microbial siderophores in enhancing virulence of T3 gonococci. The results add to the evidence that the ability to acquire iron is an important determinant of virulence.     

Virulence of transparent and opaque colony types of Neisseria gonorrhoeae for the genital tract of mice

The virulence of transparent (Tr) and opaque (Op) colony types of Neisseria gonorrhoeae in the genital tract of female mice was evaluated at two stages of oestrous. Isogenic pairs of Tr and Op variants were isolated from N. gonorrhoeae strain 57-120. Both variants exhibited a T2 morphology, but only the Op variant possessed protein II (P.II) in outer-membrane fractions. When administered by intravaginal inoculation Op gonococci were highly infective only for mice in late pro-oestrous, whereas Tr gonococci were virulent for mice at both late pro-oestrous and dioestrous. Gonococci recovered from the uterus were of both Tr and Op phenotypes in equal proportions when mice were infected at dioestrous with Tr cells. In contrast, greater than 90% of recovered colonies were of Op phenotype when mice were infected at late pro-oestrous with either Op or Tr cells. These results indicate that the virulence of gonococci for the genital tract of female mice differs from that for the chicken embryo. Furthermore, gonococcal survival in the female genital tract might be attributable to phase variation from Tr to Op phenotypes.     

Detection of latent cytomegalovirus in murine salivary and prostate explant cultures and cells.

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.     

Detection and differentiation of iron-responsive avirulent mutants on Congo red agar.

Agar medium containing Congo red dye differentiates virulent and avirulent colonies of Shigella, Vibrio cholerae, Escherichia coli, and Neisseria meningitidis. Like virulent plague bacilli, wild-type cells of these species absorb the dye and produce red colonies. Mutants or colonial variants have been isolated that fail to absorb the dye and produce colorless colonies. These mutants exhibit reduced virulence in the chicken embryo model, but their virulence is enhanced by supplementation with iron. Of those species tested, only Neisseria gonorrhoeae isolates failed to grow in the presence of this dye. Inhibition of growth by Congo red may thus provide a simple means for differentiating gonococci from other Neisseria.     

Expression of both M protein and hyaluronic acid capsule by group A streptococcal strains results in a high virulence for chicken embryos

The human pathogenic microorganismStreptococcus pyogenes can resist against phagocytic attack of human granulocytes. Streptococcal M protein and hyaluronic acid were identified as virulence factors involved in this protection. So far, no experiments have been reported which describe the contribution of both components together in one system. We used the chicken embryo as an in vivo phagocytosis model to investigate the role of both components on the virulence of streptococci. For this, isogeneic mutants of group A streptococcal strains (GAS) which lack hyaluronic acid capsule (cap⁻) or M protein (M⁻) expression were used for infection and their virulence was compared with laboratory strains which had lost their ability to produce one or both virulence factors after long-time laboratory passages on blood agar. The experiments revealed that strains producing both M protein and hyaluronic capsule were higly, virulent. Only 1–10 colonyforming units were enough to cause a 50% lethality of 12-day-old chicken embryos. Those strains lacking one of these components showed a significant decrease in virulence. Finally, strains which failed to express either hyaluronic acid or M protein showed an additional tenfold decrease in virulence. This indicates a partial contribution of both M protein and hyaluronic acid to the virulence of GAS in the chicken embryo.     

Transmission of Immunity to Neisseria Gonorrhoeae from Vaccinated Hens to Embryos

Presently, there is no practical laboratory animal model for the evaluation of experimental gonococcal vaccines. The chimpanzee has been used recently (1), but this animal is expensive and difficult to handle. On the other hand, the. susceptibility of the chick embryo to certain pathogenic microorganisms has provided an excellent model for the study of their virulence (2,3,8,9). In our laboratory, we have confirmed the original observation of Bumgarner et al (4), that Neisseria gonorrhoeae colony types T₁ and T₂ (virulent for man) are significantly more virulent for the chick embryo than T₃ and T₄ (non-virulent for man). However, chick embryo neutralization studies (4,5) have shown the unsuitability of the embryo as an animal model for the protective capacity of vaccines against gonorrhea, since there is only a ten-fold difference between the ability of normal and hyper-immune rabbit sera to protect the embryos against gonococcal challenge. Because of these limitations, we have investigated the ability of embryos obtained from Leghorn hens immunized intravenously with experimental gonococcal vaccines, to withstand a challenge with N. gonorrhoeae     

Chicken embryo lethality assay for determining the virulence of Riemerella anatipestifer isolates

Riemerella anatipestifer is the causative agent of polyserositis and septicaemia in waterfowl. Twenty-one serotypes have been reported, and there is a strong variation in virulence between strains according to serotype or strain. However, little information is available to assess virulence, such as virulence-associated genes; thus, it is difficult to estimate the risk from field strains. Hence, we established a chicken embryo lethality assay (ELA) model to determine the virulence of R. anatipestifer strains. Three virulent strains (RA T1, RA T7, and V-1) and three avirulent strains (Av-1, Av-2, and Av-3), which were confirmed by duck challenge, were used to perform the ELA. Inoculating 10² to 10⁴ colony-forming units into the allantoic cavity of 10-day-old embryos discriminated between virulent and avirulent strains based on mortality. Differences in invasion rates into embryonic tissues were found between the RA T1 and Av-1 strains. The maximum colony-forming units of the RA T1 strain were about 1000 times higher than those of the Av-1 strain in the tissue invasion rate for 4 days. We found that the virulent strains killed embryos at mortality rates ≥50% during the first 3 days after inoculation and that the avirulent strains had death rates of ≤20% over 5 days. These results obtained by repeated testing suggest that the ELA could be used as a first-line screening method to determine the virulence of R. anatipestifer strains.     

Studies on the Virulence of Neisseria gonorrhoeae I. Relation of Colonial Morphology and Resistance to Phagocytosis by Polymorphonuclear Leukocytes

Colonial varieties of Neisseria gonorrhoeae that are associated with virulence, types 1 and 2, were more resistant to phagocytosis by rabbit exudative polymorphonuclear leukocytes than colonial types of lesser virulence, types 3 and 4. Type 1 bacteria were resistant and type 4 gonococci were susceptible to phagocytosis by human polymorphonuclear leukocytes. Recent local type 1 isolates were similar in resistance to type 1 organisms of a standard laboratory strain (F62). Living and Formalin-treated, heat-killed, type 1 gonococci were equally resistant to phagocytosis. The antiphagocytic property of virulent colonial types was independent of leukotoxic action. Phagocytosis of both type 1 and type 4 gonococci by rabbit and human leukocytes was bactericidal. Rabbit leukocytes were superior to human leukocytes in killing gonococci. The results suggest that N. gonorrhoeae has virulence properties similar to those of extracellular bacterial pathogens, i.e., virulence is associated with antiphagocytic properties.     

Gonococcal color and opacity variants: virulence for chicken embryos.

Previous studies have noted that the prevalence of certain gonococcal colony types is influenced by the physiological state of the human host. Consequently eight different gonococcal strains were passed on clear typing medium, and opaque (Op) and transparent (Tr) variants of heavily pilated (P++) bacteria were selected. P++Op and P++Tr were injected into 11-day-old chicken embryos, and the resulting mortality was determined. In every case the Tr variants were more lethal to the embryos than were the Op variants (P = 0.01). P++Tr were stable in vivo, but chicken embryos injected with P++Op strains developed bacteremia with P++Tr organisms. However, chicken blood was not bactericidal for either colonial variant. These results indicate that transparent pilated bacteria may be more virulent than opaque pilated bacteria and that in vivo selection of transparent forms occurs.     

Transmission of Immunity to Neisseria Gonorrhoeae from Vaccinated Hens to Embryos

Presently, there is no practical laboratory animal model for the evaluation of experimental gonococcal vaccines. The chimpanzee has been used recently (1), but this animal is expensive and difficult to handle. On the other hand, the. susceptibility of the chick embryo to certain pathogenic microorganisms has provided an excellent model for the study of their virulence (2,3,8,9). In our laboratory, we have confirmed the original observation of Bumgarner et al (4), that Neisseria gonorrhoeae colony types T₁ and T₂ (virulent for man) are significantly more virulent for the chick embryo than T₃ and T₄ (non-virulent for man). However, chick embryo neutralization studies (4,5) have shown the unsuitability of the embryo as an animal model for the protective capacity of vaccines against gonorrhea, since there is only a ten-fold difference between the ability of normal and hyper-immune rabbit sera to protect the embryos against gonococcal challenge. Because of these limitations, we have investigated the ability of embryos obtained from Leghorn hens immunized intravenously with experimental gonococcal vaccines, to withstand a challenge with N. gonorrhoeae.     

Detection and localization of avian alphaherpesviruses in embryonic tissues following in ovo exposure

We investigated whether chicken embryonic tissues are susceptible to infection with virulent Marek’s disease virus (MDV). Groups of embryonic day (ED) 17 chicken embryos and 1-day-old chicks were compared for tissue sites of viral persistence of MDV and herpesvirus of turkeys (HVT) in lungs, thymuses, bursae of Fabricius and spleens. MDV DNA was detectable in the lungs and thymuses of embryos at 3 days post-inoculation (DPI) by in situ hybridization, while HVT DNA was only present in embryonic lungs. The target cells in lungs and thymuses appeared non-lymphoid and lymphoid, respectively. By 5 days post-inoculation, both viruses were detectable in all organs examined and persisted after hatch. Although MDV DNA was present in the embryo, there was little evidence of viral replication. These findings demonstrate the differences in pathogenesis of embryonic infection with MDV and HVT and provide evidence that the chicken embryo is susceptible to infection with a virulent avian herpesvirus.     

Chicken embryo model for type III group B beta-hemolytic streptococcal septicemia.

A lethal septicemia was induced in 11- and 12-day-old chicken embryos with intravenous inoculation of relatively small numbers of a clinical isolate (GBBHS-III-Bell) or a reference strain (GBBHS-III-D136-C) of group B beta-hemolytic streptococci (GBBHS). GBBHS-III-Bell was more virulent than GBBHS-III-D136-C, and 11-day-old chicken embryos were more susceptible than 12-day-old chicken embryos. Type-specific rabbit antisera protected the embryos from bacterial challenge, and this protective effect was absorbed with homologous but not heterologous GBBHS strains. A heterologous antiserum and normal rabbit sera provided some protection, which could be absorbed with either homologous or heterologous GBBHS strains. The chicken embryo is a suitable animal model for the study of infection and immunity with GBBHS type III.     

The chicken embryo as a model for campylobacter invasion: comparative virulence of human isolates of Campylobacter jejuni and Campylobacter coli.

Eleven-day-old chicken embryos were used to compare the relative virulence of minimally passaged human isolates of Campylobacter jejuni and Campylobacter coli. Graded doses of bacteria were inoculated onto the chorioallantoic membrane, and 50% lethal doses were calculated at 72 h postinfection. Strains varied markedly in their ability to invade the chorioallantoic membrane and kill the embryos. The 50% lethal doses varied by about 6 logs for 25 strains of C. jejuni, and by 2 logs for 5 strains of C. coli. Although both outbred and inbred embryos were employed in the study, the latter were found to be more susceptible to infection with most strains. All isolates were screened for plasmid DNA, but there was no apparent relationship between plasmid content and virulence of strains for the embryos. Neither could virulence be associated with the production of siderophores by the strains. The ability of selected strains of C. jejuni to invade the liver of embryos was also studied. The number of campylobacters culturable from the liver was found to be inversely related to the 50% lethal dose of the strain. By inoculating 11-day-old embryos intravenously, it was possible to demonstrate that a strain of C. jejuni which was poorly virulent after chorioallantoic inoculation was relatively noninvasive. Invasiveness alone, however, could not fully account for the lethality of two highly virulent strains of C. jejuni administered by the intravenous route. Finally, there was no correlation between motility and virulence in this model system.     

Propagation and assay of virulent and attenuated JMV Marek's disease-associated tumour cells

JMV tumour cells were shown to cause a lethal lymphoblastic leukaemia in young chickens as well as in chicken embryos. The incubation period was very short but dose-dependent. Chickens died in 4 to 12 days, embryos in 7 to 14 days, after inoculation. Embryo-passaged attenuated JMV (JMV-A) caused the same lesions in embryos as virulent JMV. The dose-response relationship depended on the route of inoculation and on the quality of the tumour cell preparation. Intramuscular (i.m.) inoculation of leukaemic blood or embryo lymphoblasts provided the most satisfactory response. Intraperitoneal (i.p.) inoculation and lymphoblastic chicken spleens as a source of JMV were definitely less suitable. The dose-response curves obtained in yolk sac-inoculated embryos were similar to the curves obtained by i.m. inoculation of chickens. Only 4 to 10 lymphoblasts were needed per lethal dose (50%) in chickens and 50 to 80 in embryos. The pathogenicity and antigenicity of JMV and JMV-A were strictly cell-associated. No Marek's disease (MD) virus or any other avian virus could be detected, either by various virus isolation procedures, or by serological methods. Contact transmission of JMV to other chickens did not occur. Antibodies against surface antigens on JMV lymphoblasts were detected in JMV and JMV-A chicken hyperimmune sera. These sera reacted against MD lymphoblastoid cell lines (HPRS-1 & 2, MSB-1) as well as MSB-1 anti-serum, but all sera reacted also against thymus lymphocytes from normal chickens. The results of absorption tests suggested that the surface antigens of JMV lymphoblasts and of the tested cell lines were not identical. The majority of tumour cell surface antigens appeared to represent genetically specific histocompatibility or lymphocyte antigens. A common MD tumour-associated surface antigen (MATSA) could not be identified serologically (FA test) on the tumour cells studied.     

A study on the long-term immunity induced by La Sota strain of Newcastle disease virus grown in a BS/BEK cell line of bovine embryo kidney origin.

Twenty-day-old susceptible chickens were divided into three groups; two were vaccinated with inactivated, water in oil emulsified La Sota strain of Newcastle disease virus (NDV) obtained from a bovine embryo kidney (BS/BEK) cell line and from chicken embryos, respectively. The third unvaccinated group represented the control. At 30-day intervals subgroups were exposed to the Herts 33 virulent NDV strain. Serological and clinical findings showed no appreciable difference in the immunogenicity of the antigen from either culture systems and no significant differences could be observed in its ability to protect against ND challenge.     

Role of Pili in the Virulence of Neisseria gonorrhoeae

Gonococci of the colonial types that are associated with virulence, types 1 and 2, have pili that enable the bacteria both to attach in vitro to human epithelial cells and to resist phagocytosis by polymorphonuclear leukocytes. These piliated gonococci also agglutinate various mammalian and chicken erythrocytes. Gonococci of an avirulent colonial type, i.e., type 4, have no pili and neither attach to epithelial cells or erythrocytes nor resist phagocytosis. Like the type 4 bacteria, mechanically or enzymatically (trypsin) depiliated type 1 gonococci failed to attach to epithelial cells and erythrocytes and were susceptible to phagocytosis. Pili of types 1 and 2 gonococci were antigenically similar. Both type 1 gonococci and pili isolated from them induced in rabbits antibody that (i) precipitated gonococcal pili in immunodiffusion, (ii) reacted with piliated gonococci as tested by indirect immunofluorescent analysis, (iii) inhibited attachment of piliated gonococci to both human epithelial cells and erythrocytes, and (iv) opsonized piliated gonococci.     

Virulence of Francisella spp. in chicken embryos

We examined the utility of infecting chicken embryos as a means of evaluating the virulence of different Francisella sp. strains and mutants. Infection of 7-day-old chicken embryos with a low dose of F. novicida or F. tularensis subsp. holarctica live vaccine strain (LVS) resulted in sustained growth for 6 days. Different doses of these two organisms were used to inoculate chicken embryos to determine the time to death. These experiments showed that wild-type F. novicida was at least 10,000-fold more virulent than the LVS strain. We also examined the virulence of several attenuated mutants of F. novicida, and they were found to have a wide range of virulence in chicken embryos. Fluorescent microscopic examination of infected chicken embryo organs revealed that F. tularensis grew in scattered foci of infections, and in all cases the F. tularensis appeared to be growing intracellularly. These results demonstrate that infection of 7-day-old chicken embryos can be used to evaluate the virulence of attenuated F. tularensis strains.     

Virulence of Campylobacter jejuni for chicken embryos is associated with decreased bloodstream clearance and resistance to phagocytosis.

The 11-day-old chicken embryo has been shown to be a useful animal model for comparing the virulence of human isolates of Campylobacter jejuni. Virulence in this system is associated with the ability to invade the chorioallantoic membrane and to survive and proliferate in vivo. In this study, the survival and multiplication of C. jejuni in the embryonic host was investigated. It was possible to enhance the virulence of a relatively avirulent C. jejuni strain by passaging it intravenously through the embryos. The resulting isogenic variants demonstrated enhanced abilities to survive in vivo but were still unable to invade when inoculated onto the chorioallantoic membrane. The bloodstream clearance of C. jejuni was studied, and virulent, but not avirulent, strains persisted and multiplied both in the bloodstream and in embryonic liver. Virulent strains also were cleared significantly more slowly from the bloodstream of adult BALB/c mice after intravenous challenge than were avirulent strains. C. jejuni strains which were cleared slowly in vivo were also ingested slowly in vitro by mouse peritoneal macrophages. Clearance studies in mice pretreated with cobra venom factor demonstrated that opsonization by serum complement was not a prerequisite for clearance of campylobacters from the murine bloodstream.     

Flavobacterium columnare colony types: Connection to adhesion and virulence?

Four different colony morphologies were produced by Flavobacterium columnare strains on Shieh agar plate cultures: rhizoid and flat (type 1), non-rhizoid and hard (type 2), round and soft (type 3), and irregularly shaped and soft (type 4). Colonies produced on AO agar differed from these to some extent. The colony types formed on Shieh agar were studied according to molecular characteristics [Amplified Fragment Length Polymorphism (AFLP), Automated Ribosomal Intergenic Spacer Analysis (ARISA), and whole cell protein SDS-PAGE profiles], virulence on rainbow trout fingerlings, and adhesion on polystyrene and fish gills. There were no molecular differences between colony types within one strain. Type 2 was the most adherent on polystyrene, but type 1 was the most virulent. Adhesion of F. columnare strains used in this study was not connected to virulence. From fish infected with colony type 1, three colony types (types 1, 2 and 4) were isolated. Contrary to previous studies, our results suggest that strong adhesion capacity may not be the main virulence factor of F. columnare. Colony morphology change might be caused by phase variation, and different colony types isolated from infected fish may indicate different roles of the colony morphologies in the infection process of columnaris disease.