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.

Cysticercus tenuicollis vesicle in fetal structures: report of a case.

Cysticercus tenuicollis is the larval stage of the canine tapeworm Taenia hydatigena, the presence of which has been reported in wild and domestic ruminants all over the world. It is a common parasite of small ruminants in the north of Portugal. C. tenuicollis is generally seen attached to the omenta, the mesenteries or also found in the liver. In the ewe, tissue lesions have been associated with degenerative cysts or with oncosphere migrations. Unusual locations of the cysticerci of T. hydatigena have been described. The most frequent unusual locations are in the lungs, the kidneys and the brain. Less common locations have been reported to occur in the ovaries, uterine tubes, uterus, cervix and vagina. In the case described here, and for the first time, an aberrant location of a C. tenuicollis vesicle was found inside the chorion-allantoic membrane of a goat's foetus, in a gemelar gestation of approximately 70 days. Finding a C. tenuicollis vesicle inside fetal membranes forewarns of the possibility of larval migrations into the fetal structures during pregnancy, which is particularly concerning in human populations that are infested.

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.

Persistence of Tritrichomonas foetus in naturally infected cows and heifers in Argentina.

Tritrichomonas foetus infection was investigated in 76 pregnant and 64 non-pregnant cows slaughtered in the local abbattoir and in two different lots of first-service heifers that were found to be non-pregnant 60 days post breeding (PB). In live and slaughtered animals, mucus samples were obtained from the vagina and from the vagina and uterus, respectively, using a "screw-head scraper rod". In pregnant cows, samples of amniotic and allantoid fluid were also collected, as well as samples from the stomach contents of the fetuses. All samples were cultured in Modified Plastridge Medium. T. foetus was isolated from three pregnant and two non-pregnant slaughtered cows. Parasites were recovered from the vagina of these five cows, as well as from the uterus in two cases and from the fetus in one case. Lot I of first-service heifers consisted of 323 females from eight different farms. Bulls infected with T. foetus from these farms were culled or treated, and heifers found empty at diagnosis of pregnancy were culled. Lot II consisted of 120 heifers from a single farm where T. foetus was controlled only in bulls. All heifers from Lot I were T. foetus negative. In Lot II, 12 of 120 heifers (10%) were T. foetus positive. In ten of these the parasites were observed once, in one at 60 days PB, in seven at 160 days PB and in two at 240 days PB; in the remaining two infected heifers, an irregular pattern of isolation persisted during 300 days PB. On the basis of these results, control methods are discussed and analysed.

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.

Growth, development, pathogenicity, and transplantation of Echinostoma caproni (Trematoda) on the chick chorioallantois.

Excysted metacercariae of Echinostoma caproni were cultivated on the chorioallantoic membrane (CAM) of 6-day-old domestic chick embryos for 2-13 days postinoculation. There was no significant difference in the body area of fixed and stained preovigerous worms from the CAM versus those grown in domestic chicks. However, ovigerous worms from the CAM were significantly smaller than those from chicks. Worm development, i.e., gonadal differentiation, uterine curling, vitellinogenesis, ovigerousness, and oviposition, took 1 day longer on the CAM than in the chick. Histopathologic studies of worms attached to the CAM were done on cryostat and paraffin sections stained with hematoxylin and eosin. Some worms attached to the CAM by their collar spines and acetabulum, whereas others penetrated the chorionic epithelium and encapsulated in the mesenchyme. Pathogenicity to the CAM included hyperplasia of the chorionic epithelium, hemorrhagia, reduced fibrocytes and blood vessels, but increased lymphocytes and eosinophils in the mesenchyme. Attempts to transplant 11-day-old CAM worms to new CAMs were unsuccessful.

Cultivation of excysted metacercariae of Echinostoma caproni (Trematoda) to ovigerous adults on the chick chorioallantois.

Excysted metacercariae of Echinostoma caproni were cultivated on the chick chorioallantoic membrane (CAM) maintained at 38.5 +/- 1 C and a relative humidity of 60-65%. Of 59 6-day-old embryos, each inoculated with 25 metacercariae, 29 (49.2%) were infected 2-12 days postinoculation. The total number of worms recovered from the infected eggs was 163 or 22.5% of the 725 inoculated metacercariae. Eggs contained from 1 to 12 (average 5.6) worms per CAM. Worm length increased rapidly from an average of 0.5 mm at 2 days to about 3.0 mm at 6 days postinoculation. Ovigerous worms first were seen on day 8 PI, but fluke eggs did not develop embryos. Worm development in ovo lagged about 1 day behind that of in vivo worms. One worm maintained for 17 days on 2 successive CAMs reached 6 mm in length, contained about 100 eggs in its uterus, and laid an additional 100 eggs on the CAM surface.

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)

Comparative growth and development of the metacercariae of Fibricola seoulensis (Trematoda: Diplostomidae) in vitro, in vivo and on the chick chorioallantois.

The growth and development of the metacercariae of F. seoulensis cultivated in vitro or on the chick chorioallantois were assessed by comparison with the optimum process of maturation in albino rats and new born chickens. The process of maturation was divided for convenience into six stages: Stage 1; cell multiplication, Stage 2; body shaping, Stage 3; separation of genital anlagen, Stage 4; organogeny, Stage 5; gametogony, and Stage 6; oviposition. In Hank's and Tyrode's solutions, the metacercariae were alive up to 200 days or more at 4 degrees C without any development. The in vivo maturation process in rats or chicks was as follows: stage 1 from 6 hours; stage 2 from 24 hours; stage 3 from 48 to 72 hours; stage 4 from 3 to 4 days; stage 5 from 4 to 5 days; and stage 6 from 5 to 8 days. Despite unsuccessful infection of the metacercariae to 12 day old chicks, fully mature worms of stage 5 or 6 were recovered from new born chicks (1 to 2 days old). The metacercariae of F. seoulensis grown in vitro were up to stage 3 and no further maturation was observed. Of various media employed, the medium NCTC 109 (Gibco) or NCTC 135 (Gibco) supplemented with 20% egg yolk or 20% whole egg macerate or 0.5% yeast was basically required for the earlier development of the fluke. It took 16.1 days (in average) to reach the stage 3 after cultivation. The metacercariae cultivated on the chorioallantoic membranes of 6-13 day old chick embryo at 37-38 degrees C showed their full development up to stage 5 or 6. However, the worms were in general remarkably retarded, compared with those grown in rats or chickens. In the experiments of worm transplant, although the transfer was failed from in vitro culture to in vivo of rats (per os), the transplants from in vitro culture to the chorioallantois and from the chorioallantois to in vivo of rat host were successful with or without development of the transferred worms. In the present study, it was observed that the metacercariae of F. seoulensis can be maintained in vitro media with poor development as well as fully matured in 1 to 2 day-old chicks or on the chorioallantois at a very low rate.

An alternative method for the culture of Diplostomum spathaceum (Trematoda) in chick embryos.

Metacercariae of Diplostomum spathaceum were maintained in the allantoic cavities of chick embryos for 15 days. Some embryos had 0.2 ml chicken serum added to the allantoic cavity each day. Although the level of development varied considerably, worms from embryos with added serum developed hind bodies that were substantially larger than those of parasites maintained without added serum. There was no evidence that any worm ingested blood, and only 1 individual, from the serum-augmented group, became ovigerous.

Site-finding and pairing of Echinostoma revolutum (Trematoda) on the chick chorioallantois.

Site-finding of 14-day-old Echinostoma revolutum from the domestic chick was studied by inoculating single worms into various sites on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. Regardless of the site of inoculation, single worms were attracted significantly to the area of the CAM above the embryo. More worms were found in this site at 24 than at 1 hr postinoculation. Worm-pairing was studied in chick embryos by inoculating 2 worms in separate windows, 2 cm apart. Worm-pairing, i.e., worms in contact or within 5 mm of each other, was very evident at 24 hr. The percentage of paired worms on the CAM above the embryo was considerably less than single worms.

Growth and feeding of Echinostoma revolutum on the chick chorioallantois and in the domestic chick.

Chemically excysted metacercariae of Echinostoma revolutum cultivated on the chick chorioallantois grew slowly until day 5, more rapidly between 5 and 7 days, and slowly between 7 and 10 days. Worms did not become ovigerous in this site by 12 days, at which time studies were terminated. In contrast, chemically excysted metacercariae reared in the domestic chick were ovigerous by day 9, at which time their mean body area was about 4 times greater than the largest chorioallantoic worms. Histochemical studies, solubility tests for hematin, and X-ray microanalysis of cecal contents showed that chorioallantoic-worms fed on blood from the vascular membrane, whereas chick-worms fed on host intestinal mucosa.

The culture of Diplostomum spathaceum metacercariae on the chick chorioallantois.

Metacercariae (2,828) obtained from the lens of naturally infected Aplodinotus grunniens were transplanted onto chorioallantoic membranes (CAM's) of 94 eggs (means 30/egg) 5 to 12 days old. Membranes were examined 2 to 8 days later and 381 flukes were recovered. Two hundred fifty-nine chorioallantoic-grown worms were transferred to 28 additional embryos 3 to 8 days after the initial inoculation. These eggs were examined 3 to 11 days later. Fifty-three serially transferred worms were recovered after 7 to 14 days on CAM's. Six stages of development were recognized: stage 1, immature; stage 2, genital rudiment; stage 3, testes; stage 4, follicular ovary; stage 5, vitellaria; stage 6, ovigerous. The intestinal ceca of many worms (except stage 1) contained ingested blood. Trematode eggs oviposited on CAM's were embryonated in tap water, and viable miracidia were observed in 15. No previous study has achieved gonadal development in Diplostomum spathaceum on the CAM. Furthermore, inasmuch as metacercaria matured and produced fertile eggs, this form is shown to be a useful model for the study of trematode differentiation. The development of metacercariae to ovigerous adults capable of producing viable miracidia suggests that the CAM shares characteristics with the intestine of the definitive host, a piscivorous bird.

Complete development of Isopora suis of swine in chicken embryos.

Development of the swine coccidium , Isospora suis, in embryonated chicken eggs is described. The allantoic cavities of eight-to-ten-day-old white Leghorn embryos were inoculated with either 100,000 or 200,000 sporozoites. Developmental stages morphologically similar to those found in the intestines of piglets were present in the endodermal layer of the chorioallantoic membrane (CAM), beginning three days post inoculation (PI). No stages were found in the mesodermal or ectodermal layers of the CAM and none were observed in heart, lung, liver, or spleen. Type I meronts and merozoites were found on days 3 through 10 PI. Type II meronts and merozoites were found days 4 through 10 PI. Mature microgamonts , macrogamonts, and oocysts were found on days 7 through 10 PI. Oocysts appeared to be retained in the endodermal cells and in ovo sporulation did not occur. Attempts to sporulate CAM-derived oocysts were not successful. Isospora suis was not pathogenic for embryos under the conditions of this study. This study represents the first fully documented report of complete development of a mammalian coccidium in chicken embryos.

Growth, development and pairing of Leucochloridiomorpha constantiae (Trematoda) metacercariae on the chorio-allantois of chick embryos cultivated in vitro.

A simple in vitro technique was devised to culture chick embryos in Petri dishes from the 4th to the 21st day of incubation. Leucochloridiomorpha constantiae (Trematoda) metacercariae were placed either singly or multiply (5/embryo) on the chorio-allantois of in vitro grown embryos on day 7 and were removed on day 14. Growth and development studies were also made on worms grown singly or multiply (5/chick) in the bursa of Fabricius of the domestic chick. Worms grown singly or multiply in embryos were sexually mature, although eggs from these worms were abnormal when compared with eggs from worms recovered from chicks. The mean body area of worms from chicks was 2-3 times greater than that of worms from embryos. The mean body area of single worms from embryos was significantly larger than that of worms grown multiply in this site. However, the mean body area of multiple worms from the chick was significantly larger than that of single worms from this site. Worm pairs or clusters were seen in all embryos with the multiple infections.