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

Out of the 14 avian ß-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian ß-defensin 11 (Gga-AvBD11). Given its specific localization and its established antibacterial activity, Gga-AvBD11 appears to play a protective role in embryonic development. Gga-AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to ß-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed ß-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-ß-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga-AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella, and avian influenza virus. Gga-AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga-AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.

An in vitro model of infection of chicken embryos by Cryptosporidium baileyi.

Cryptosporidiosis is one of the most prevalent parasitic infections in domesticated, caged and wild birds. Cryptosporidium baileyi is the most common species reported in a wide range of avian hosts. Although this parasite is well investigated, there is no adequate in vitro model for its endogenous development, and therefore, knowledge of each life cycle phase is scarce. In the present study, an in vitro model for C. baileyi in chicken embryos was developed and the complete life cycle investigated by light and electron microscopy, including both the sexual and asexual reproduction stages. The complete life cycle of C. baileyi was observed during 1-96 h post inoculation (PI), and the average reproduction number of C. baileyi oocysts in allantoic fluid of each chicken embryo was greatest at 168 h PI. These results suggest that chicken embryos could adequately represent the natural host cells and support the development of all the endogenous life cycle stages of C. baileyi, and also provide a new and effective in vitro cultivation system for further studies on antigens, virulence, infectivity, metabolites, and sensitivity of drugs against parasites.

Establishment of Eimeria tenella (local isolate) in chicken embryos.

Development of an in vitro Eimeria (E.) tenella model could be valuable as a tool for vaccine, coccidiostats or molecular biology research. 1.0 × 10,000 sporozoites per 0.1 mL were inoculated into the allantoic cavity of ten-day-old chicken embryos. The complete life-cycle of E. tenella was accomplished in eight-nine days at 37 °C and 70% humidity. The addition of 100 U insulin to the embryos could remarkably improve the output of oocysts. The development of the parasite within the embryos was systematically observed, allowing guidelines to be set regarding the appropriate times at which different developmental stages of the parasite may be sampled.

Immunohistochemical and polymerase chain reaction studies in Neospora caninum experimentally infected broiler chicken embryonated eggs.

The diagnostic characteristics of immunohistochemistry (IHC) and polymerase chain reaction (PCR) methods were studied in the tissues of broiler chicken embryos experimentally infected by Neospora caninum. An infection with N. caninum NC-1 isolate was conducted in 70 broiler chicken embryonated eggs randomly divided into seven equal groups. After 8 days of incubation, six groups were inoculated with 10, 10(2), 10(3), 10(4), 10(5), and 10(6) doses of tachyzoites/embryonated egg. The 7th group was considered as control. The mortality rate and pathological changes of the dead embryos and hatched chickens up to 60 days old were noticed. Consecutive sections to those used for histopathological examination including the liver, heart, brain, and chorioalantoic (CA) membrane were subjected to IHC. The intensity and distribution of the immunostaining was graded as highly to mildly positive. For PCR procedure, DNA was extracted from 50mg of the tissues and primer pair Np21/Np6 was used for amplification of the Nc-5 gene. The results of the immunosignaling ranged from variable degrees of mild to moderate staining as dark-brown to brown and coarsely to finely granular, mostly within the cytoplasm of infected cells such as the endothelial cells of blood vessels. The parasite aggregation was more predominant in the heart than other tissues. Immunoreactivity for N. caninum antigen was multifocally moderate positive in the heart, liver and CA of the 10(3) dose, and also heart, liver, brain and CA of the 10(4) dose. IHC showed mildly positive in the liver and heart of the chicken embryos infected with 10 and 10(2) tachyzoites, as well. The results of the PCR confirmed the existence of the parasite in all of the examined tissues from the 10(3) and 10(4) doses. In conclusion, the results indicate a good agreement between IHC and PCR in diagnosis of neospora antigen in the infected tissues.

Chick embryo tracheal organ: a new and effective in vitro culture model for Cryptosporidium baileyi.

In the present study, chick embryo tracheal organ (TOCs) was used to cultivate oocysts or sporozoites of Cryptosporidium baileyi. Approximately 5 × 10(4) sporozoites and oocysts mixture for group I; 5 × 10(5), 1 × 10(6), 2 × 10(6) purified sporozoites for group II, group III and group IV, respectively, were inoculated into respective chick embryo tracheal rings maintained in RPMI 1640 supplemented with 5% heat-inactivated FBS, and cultivated in each well of the 24-well culture plate at 40°C and 5% CO(2). The tracheal rings in four experimental groups (I-IV) were successfully infected with C. baileyi, and different stages of parasites were also observed under light and electron microscopy. Parasite infection and cytological alterations were noted as early as PI 72 h. The Cryptosporidium were seen attached to the edge of the tracheal epithelium, with more number of parasites in group I than that in group II, group III and group IV. The moderate nuclear swelling and chromatin margination were also detected, and the normal vertical orientation and basilar location of the nuclei of the epithelial cells were almost lost. C. baileyi that has been passed by TOCs exhibited similar immunity and molecular features with parasites before intratracheal inoculation. These results suggest that chick embryo tracheal organ is a new and effective in vitro culture model for C. baileyi and other respiratory pathogens.

Strain-dependent host transcriptional responses to Toxoplasma infection are largely conserved in mammalian and avian hosts.

Toxoplasma gondii has a remarkable ability to infect an enormous variety of mammalian and avian species. Given this, it is surprising that three strains (Types I/II/III) account for the majority of isolates from Europe/North America. The selective pressures that have driven the emergence of these particular strains, however, remain enigmatic. We hypothesized that strain selection might be partially driven by adaptation of strains for mammalian versus avian hosts. To test this, we examine in vitro, strain-dependent host responses in fibroblasts of a representative avian host, the chicken (Gallus gallus). Using gene expression profiling of infected chicken embryonic fibroblasts and pathway analysis to assess host response, we show here that chicken cells respond with distinct transcriptional profiles upon infection with Type II versus III strains that are reminiscent of profiles observed in mammalian cells. To identify the parasite drivers of these differences, chicken fibroblasts were infected with individual F1 progeny of a Type II x III cross and host gene expression was assessed for each by microarray. QTL mapping of transcriptional differences suggested, and deletion strains confirmed, that, as in mammalian cells, the polymorphic rhoptry kinase ROP16 is the major driver of strain-specific responses. We originally hypothesized that comparing avian versus mammalian host response might reveal an inversion in parasite strain-dependent phenotypes; specifically, for polymorphic effectors like ROP16, we hypothesized that the allele with most activity in mammalian cells might be less active in avian cells. Instead, we found that activity of ROP16 alleles appears to be conserved across host species; moreover, additional parasite loci that were previously mapped for strain-specific effects on mammalian response showed similar strain-specific effects in chicken cells. These results indicate that if different hosts select for different parasite genotypes, the selection operates downstream of the signaling occurring during the beginning of the host's immune response.

Exoerythrocytic development of Plasmodium ga llinaceum in primary fibroblast culture of chicken embryo / Desenvolvimento exoeritrocítico de Plasmodium gallinaceum em cultura primária de fibroblasto de embrião de galinha

In this study we assessed the susceptibility of primary fibroblast culture of chicken embryo to infection of P. gallinaceum sporozoites as well as the initial development of exoerithrocytic stages. Fibroblasts were obtained from the chest muscles of chicken embryos and sporozoites were obtained from experimentally infected Aedes fluviatilis salivary glands. After 1h, 3h, 24h, 48h and 72h periods pos-infection, cell cultures were fixed and analyzed both by indirect immunofluorescent-antibody test with anti-circumsporozoite protein monoclonal antibodies and by transmission electron microscopy. Circumsporozoite protein was detected in all parasitic forms. The mean percentage of fibroblasts with adhered or penetrated sporozoites did not significantly increase proportionately to the concentration of parasites in the inoculum, and independently if fetal calf or normal chicken sera were used in the culture medium. It was noted that the longer the incubation time, higher the possibility of the sporozoites to adhere and penetrate to fibroblats. Spozoites were observed penetrating in the fibroblast after 3h incubation when 0.68% of the cells had adhered parasites. Differentiation and development of the exoerythrocytic forms was observed after 24h incubation, when an average of 0,14% of the parasites have already invaded the cells. Developing parasites were found until 72h, when only 0.04% of fibroblasts were infected. Fibroblast cell culture seems to be a valuable experimental tool for in vitro investigation of the exoerytrocytic cycle of P. gallinaceum.

No presente estudo, avaliamos a susceptibilidade de cultura primária de fibroblastos de embrião de galinha à infecção por esporozoítas de P. gallinaceum, assim como o desenvolvimento de estágios do ciclo exoeritrocítico. Fibroblastos foram obtidos a partir da musculatura do peito de embriões de galinha e esporozoítas foram obtidos de glândulas salivares de Aedes fluviatilis experimentalmente infectados. Após períodos de 1h, 3h, 24h, 48h e 72h após a infecção, culturas de células foram fixadas e analisadas através de imunofluorescência indireta empregando-se anticorpos monoclonais contra a proteína circum-esporozoíta e microscopia eletrônica de transmissão. Proteína circum-esporozoíta foi detectada em todas as formas parasitárias. O percentual médio de fibroblastos com esporozoítas aderidos ou já penetrados não aumentou proporcionalmente com a concentração de parasitos no inóculo e independeu se o soro utilizado no cultivo celular era soro bovino fetal ou soro de galinha normal. Foi observado que, quando maior é o período de incubação, maior é a possibilidade dos esporozoítas aderirem e penetrarem nos fibroblastos. Esporozoítas foram observados penetrando em fibroblastos depois de 3h de incubação, quando 0,68% das células tinham parasitos aderidos. A diferenciação e o desenvolvimento das formas exoeritrocíticas foram observados após 24h de incubação, quando somente 0.04% dos fibroblastos achavam-se infectados. A cultura primária de fibroblastos de galinha parece ser um valioso modelo experimental para a investigação in vitro do ciclo exoeritrocítico do P. gallinaceum.

Neospora caninum infection in birds: experimental infections in chicken and embryonated eggs.

Neospora caninum causes economical impact in cattle-raising farms since it is implicated as the major cause of bovine abortions. Although infection by the parasite has been widely described in mammals, the role of birds in its life-cycle is still obscure. Therefore, this work aimed to evaluate the infection by N. caninum in different chicken models. Experimental infections were conducted in 7-day-old chicks, laying hens and embryonated eggs, where samples were analysed for parasite burden, IgG antibodies and lesions promoted. Chickens demonstrated an asymptomatic infection, although with seroconversion and systemic replication of the parasite. In laying hens, no signs of vertical transmission were observed. However, embryonated eggs inoculated by the allantoic cavity route demonstrated susceptibility to infection, with mortality rates around 50% independent of the inoculum dose. Additionally, dogs became infected after ingestion of different amounts of inoculated eggs, producing either oocysts or specific IgG antibodies. The results herein presented demonstrate that chickens may be intermediate hosts of N. caninum and that embryonated eggs could be a useful model to study the parasite's biology.

Developmental abnormalities in chicken embryos after injection proteolysis inhibitors from Ascaris suum.

It has been found that trypsin and alpha-chymotrypsin inhibitors isolated from Ascaris suum act embryotoxically and teratogenically on White Leghorn chicken embryos. Mortality rate for the chicken embryos on day 15 of incubation was 45.0 +/- 3.5% after injection of trypsin inhibitor and 44.0 +/- 3.5% after adminstration alpha-chymotrypsin inhibitor. Gross examination of surviving embryos and their dissection revealed pathological changes (abdominal dropsy, umbilical hernia, subcutaneous oedema, hemoperitoneum, hemopericardium), symptoms indicating retardation in growth (lack of down, retarded ossification of long bones, decreased mean body weight) as well as malformations (schistocelia, micrognathia, cyclopia, crossed beak, cranial deformities) after injection of inhibitors from Ascaris. The highest incidence of embryos with pathological changes and malformations was found after administration of alpha-chymotrypsin inhibitor. The most commonly occurring abnormality was schistocelia (21.4 +/- 3.88%). Growth malformations were not found in the control groups. The trypsin and alpha-chymotrypsin inhibitors present in Ascaris homogenate have a significant disturbing effect on the development of the chicken embryo.

Use of an in ovo technique to provide adults of Levinseniella sp. no. 17 that were identified as Levinseniella minuta (Trematoda: Microphallidae).

Metacercarial cysts identified as Levinseniella sp. no. 17 (Deblock, 1980) were found in the mud snail Hydrobia ulvae. These larval trematodes had never been allied to a known species of adult worm. They were chemically excysted and cultured for 120 hr on the chorioallantoic membranes of chicken embryos. The resulting adult worms were observed by light and scanning electron microscopy and details of their anatomy and topography were compiled. Their morphometric measurements were compared with those of adult worms taken from the scientific literature. A very high degree of similarity was discovered between anatomical details of the worms produced in this study and those provided for Levinseniella minuta. It was concluded that Levinseniella sp. no. 17 is the larval stage of L. minuta.

Cultivation of excysted metacercariae of Echinostoma caproni to ovigerous adults in the allantois of the chick embryo.

Chemically excysted metacercariae of Echinostoma caproni inoculated into the allantois of domestic chick embryos became ovigerous in that site within 9 days postinoculation. The egg preparation technique of Saville and Irwin was markedly better than that of a modified Zwilling procedure for obtaining large numbers of postinoculation embryos with worm infections. Adults of E. caproni from the allantois were larger and became ovigerous sooner than worms grown on the chorioallantois. Only worms from the allantois produced eggs with fully developed miracidia. Miracidia were released from these eggs, but an insufficient number was available to attempt infections in Biomphalaria glabrata snails.

[The effect of Ascaris lumbricoides suis and its trypsin inhibitor on the chick embryo]. / Dzialanie homogenatu Ascaris lumbricoides suis oraz jej inhibitora trypsyny na zarodek kury.

It has been found that tegument homogenate of Ascaris lumbricoides suis and also trypsin inhibitor isolated from it induce the Leghorn chick embryos mortality when injected into their yolk sac on 4th, 8th or 13th day of incubation. The trypsin inhibitor is one of important components of Ascaris homogenate causing mortality. There is linear interrelationship between the logarithm of dose of homogenate or trypsin inhibitor and the mortality of chickens in %. A significant decrease of mean mass of chicks injected with Ascaris homogenate or trypsin inhibitor in comparison with control groups was observed. There was more frequent occurrence of developmental abnormalities and pathological changes in groups of hatched chicks which received Ascaris homogenate or inhibitor.

In ovo cultivation of Microphallus primas (Trematoda: Microphallidae) metacercariae to ovigerous adults and the establishment of the life-cycle in the laboratory.

This investigation reports for the first time the establishment of a trematode life-cycle in the laboratory using in ovo cultivation of the adult stage. Microphallus primas metacercarial cysts were removed from Carcinus maenas crabs and chemically excysted. Using a modification of the technique developed by Fried (1962) the released metacercariae were grown in fertile hen's eggs to ovigerous adult flukes. These were removed from the under surface of the chorio-allantoic membrane of chick embryos 5 days post-infection and the fluke eggs recovered were used to infect laboratory-reared Hydrobia ulvae snails. When challenged against parasite-free crabs the cercariae released from the experimentally infected snails gave rise to metacercarial cysts that were again identified as M. primas.

Cultivation of helminths in chick embryos.

A total of 23 species from 14 families of the Digenea have been studied in chick embryos, mainly on the chick chorioallantoic membrane (CAM). Most species for which cultivation has produced ovigerous adults in chick embryos have been avian digeneans with progenetic metacercariae. Less success has been obtained with the hermaphroditic, non-progenetic, economically important trematodes such as fasciolids and echinostomatids, although post-metacercarial development has been achieved for Fasciola hepatica and Echinostoma trivolvis (synonym of E. revolutum) (see Fried and Butler, 1979; Fried and Pentz, 1983). Success with human or animal blood flukes has been minimal, although adults of Schistosoma mansoni and Spirorchis spp. (turtle blood flukes) have at least been maintained on the CAM (see Fried et al., 1982; Fried and Tornwall, 1969). Schistosome cercariae and in vitro transformed cercariae (schistosomules) should be tested in chick embryos. Marine avian schistosomes in the genera Austrobilharzia and Ornithobilharzia, along with the freshwater avian schistosome Trichobilharzia, would provide useful material for avian embryo studies on non-human schistosomes. Studies on trematodes in chick embryos have been done mainly to gain basic biological information on these parasites. That is, to identify species for which definitive hosts are not available; for studies on worm-intraspecific variation, growth and development; for studies on worm feeding and digestion; and for studies on worm-mediated chemoattraction and worm site location on the CAM (Fried, 1989). Sites other than the upper surface of the CAM have not been well-explored for digeneans, although Irwin and Saville (1988a,b) have examined the subchorioallantois and allantois as habitats for stigeids and microphallids. They have also studied the effects of serum supplements to the embryo on the enhancement of worm growth and development. Irwin and Saville's work should be extended to other helminths. The albumen in the hen's egg is a good site for the development of Clinostomum marginatum and Amblosoma sawaense (see Larson and Uglem, 1990; Fried et al., 1981), but the reasons for the better growth of these parasites in the albumen than on the CAM are not known. The inoculation of trematode larvae into CAM blood vessels, the yolk sac, the amnion and the embryo proper have not been explored and may provide useful avenues of research. Only a single study has been done with a monogenean trematode, Polystomoides sp. (see Fried, 1965), in which worms were grown on the CAM at 30 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of anticoccidial drugs in chicken embryos.

Infections of Eimeria tenella in chicken embryos were used to compare the anticoccidial activity of ten drugs. The minimal inhibitory concentration (MIC) and minimal toxic concentration (MTC) were affected by the time of inoculation into the embryos and by the chemical nature of the compounds. Some compounds (nicarbazin, amprolium) had no effect on the development of coccidia when they were injected into embryos after the day of infection. Drugs that act early in the life cycle of coccidia (robenidine, clopidol, decoquinate, diclazuril, halofuginone, monensin, salinomycin, and lasalocid) were active at 5-125 micrograms/embryo when they were injected on the day of infection. The ionophores and halofuginone were highly toxic to embryos; most synthetic compounds were nontoxic. The incubation of merozoites in drug suspensions prior to the infection of embryos did not result in embryo toxicity, but the resultant MICs were much higher than those obtained when drugs were injected directly into the embryos. Several products were essentially inactive. Neither nicarbazin nor amprolium prevented oocyst formation. The widely divergent endpoints for the MIC and MTC of anticoccidials in embryos seriously limits the application of this technique as a screen for anticoccidial drugs.

A new method for purification of Eimeria tenella merozoites.

A rapid and simple method for purifying second generation merozoites of Eimeria tenella was developed using a host tissue digestion fluid, containing 0.25% trypsin and 0.5% taurodeoxycholic acid, to liberate merozoites grown in chick embryos or from parasitized ceca. After filtration, the digestion procedure yielded 1.4 x 10(7) or 8.33 x 10(7) merozoites per embryo or cecum, respectively. These yields were nine-fold for embryos and three-fold for ceca in comparison to previous reports. Viability of the merozoites was normal as assessed by their ability to reinfect embryos and cell cultures. The new method has advantages in that large numbers of pure, viable merozoites can be obtained quickly and easily, and the procedures require minimal effort and supplies.