Granuloma formation to Capillaria hepatica eggs. I. Descriptive definition

The course of the cellular response in the liver to nonembryonated Capillaria hepatica (Bancroft, 1893) eggs given by intravenous injection into the portal circulation of unsensitized and sensitized mice was studied qualitatively and quantitatively. A gradual infiltration of predominantly mononuclear cells occurred around the eggs in the liver, leading to the formation of distinct granulomatous lesions characterized by macrophages and lymphocytes. This was followed by an infiltration of eosinophils. Previous intraperitoneal sensitization led to an earlier and an enhanced reaction to an intravenous challenge with eggs. Specificity of the cellular response was suggested by the lack of an enhanced reaction to presensitization with eggs of a closely related species, Trichuris muris. These studies indicate that granuloma formation to C. hepatica eggs has an immunological basis and furthermore the cell composition of the granuloma would suggest that a cell-mediated component may be involved as part of the specific response.     

Copillaria hepatica: Fine structure of egg shell

The structure of the Capillaria hepatica egg shell was studied with the electron microscope and correlated with light microscope histochemical observations. The shell is composed of fibrous and nonfibrous components, both of which stain for protein. The fibrous component, the major portion of the shell, consists of submicroscopic fibers. The nonfibrous component is located in the outer region of the shell but is not always visible; when present it has a reticulated appearance in electron micrographs. The fibrous component is divided into outer and inner regions. The outer region is composed of radially arranged pillars which are connected at their outer surface by a beam-like network and are anchored at the base to a compact inner region. The inner region consists of a series of concentrically arranged lamellae above which is located a nonlaminated region where the pillar bases originate. At each polar end of the shell is a single opening plugged with a material which contains acid mucopolysaccharide. The fine structure of the body of the plug is unresolvable with the electron microscope; its outer surface is impregnated with electron dense particles. Externally the shell is covered by a 250 Å thick continuous membrane which is in close opposition to the surrounding host tissue.     

The First Case of Capillaria hepatica Infection in a Nutria (Myocastor coypus) in Korea

This study reports the first case of Capillaria hepatica infection in a nutria in Korea. Ten nutrias, captured near the Nakdong River, were submitted to our laboratory for necropsy. White-yellowish nodules were found in the liver of 1 of the nutrias at necropsy. Histologically, the lesions were granulomatous, and infiltrations of lipid-laden macrophages, eosinophils, and several multinucleated giant cells were observed. The lesions consisted of numerous eggs and necrotic hepatocytes. The eggs were lemon-shaped and had polar plugs at the ends of both long sides. The eggs were morphologically identified as those of C. hepatica. Worldwide, C. hepatica infection in nutrias is very rare. Nutrias are a kind of livestock, as well as wildlife; therefore, an epidemiological study for parasitic infections needs to be conducted.     

Granulomatous hypersensitivity and antibody production in response to antigens of Capillaria hepatica eggs

Soluble egg antigens of Capillaria hepatica were fractionated by gel filtration chromatography into two major protein peaks of 300,000 and 14,000 mol.wt. The unfractionated antigen and both peaks were capable of sensitizing mice to produce quantitatively larger liver granulomas upon experimental innoculation of eggs; the increase in granuloma size was related to the amount of antigen used to sensitize. The antibody response in C. hepatica infected mice was primarily directed toward the high molecular weight components. Comparison of these findings with those reported for the Schistosoma mansoni egg granuloma suggests a diversity in the mechanisms of granuloma formation among helminth parasites.     

In vitro cultivation of Fasciola hepatica metacercariae and of partially developed flukes recovered from mice

Attempts were made to culture the metacercariae of Fasciola hepatica under a wide variety of conditions. Of the media tested, the most successful was NCTC 135 plus 50% heat inactivated chick serum and sheep red blood cells at 37°–38°C. In this medium, somatic development of newly excysted juveniles was similar to that of flukes recovered from the liver of a mouse 11 days post-infection. There was, however, no corresponding development of the genital rudiment. Various supplements, such as liver extract, bile, yeast extract, embryo extract, egg products, monolayer cells and diphasic media were tested, but none enhanced development. The effects of various physical parameters on growth and development in vitro were examined. Cultured metacercariae appeared to be in a state of ‘suspended animation’; when injected intraperitoneally into mice they developed into egg-producing adults. Flukes recovered from the abdomen and liver of mice continued their somatic growth in vitro but their genitalia failed to develop further.     

Leucine Aminopeptidase in Eggs of the Soybean Cyst Nematode Heterodera glycines

Supernatant from a sonicated macerate of eggs of Heterodera glycines hydrolyzed L-leucine β-naphthylamide and L-leucine 7-amido-4-methylcoumarin. Rate of substrate hydrolysis was influenced by pH and increased with the duration of incubation. A Michaelis-Menten constant of 0.15 mM was obtained. Rate of substrate hydrolysis was decreased by freezing egg supernatant for 26 days or heating above 60 C for 5 minutes. When egg supernatant was incubated with six different substrates, L-leucine β-naphthylamide was hydrolyzed most readily and L-valine β-naphthylamide the least readily. The rate of substrate hydrolysis by egg supernatant was not increased by pretreatment of eggs with 3 mM zinc chloride for up to 14 days.     

Does the Polyphagous Egg Parasitoid <Emphasis Type="Italic">Trichogramma platneri</Emphasis> Nagarkatti (Hymenoptera: Trichogrammatidae) Display Behavioral Plasticity When Parasitizing Different Hosts?

Trichogramma platneri oviposition behavior on Amorbia cuneana egg masses was investigated under laboratory conditions. No relationship was detected between host surface area, number of edge turning on host eggs, and parasitoid clutch size. It was also observed that females parasitized the egg mass randomly by drilling into the egg mass and parasitizing individual eggs without using stereotypical behaviors to assess individual hosts (i.e. drumming and turning). However, clutch size did change due to ovipositional experience as naïve wasps with little or no ovipositional experience (<6 eggs parasitized), allocated significantly more progeny per host than wasps with longer ovipositional experience (24-h oviposition experience on a single egg mass). Moreover, naïve wasps parasitized significantly more eggs on the outer edge of the egg mass than experienced wasps. We contend that the physical characteristics of A. cuneana eggs and egg masses preclude T. platneri from completely discriminating between individual eggs. However, because T. platneri may be using kairomones from the egg mass, this described oviposition strategy remains effective.     

Hymenolepis nana: Maturation in an immunosuppressed unnatural rat host

When eggs of the dwarf tapeworm Hymenolepis nana, cycled exclusively and directly through mice for more than 10 years, were inoculated into previously uninfected inbred Fischer (F344) strain rats, they failed to mature in the rat intestinal lumen. Eggs of H. nana inoculated into the rat developed normally into cysticercoids (cysts) in the intestinal tissue, but thereafter failed to mature in the lumen except when the host was treated with cortisone acetate from the day of cyst maturation. The Fischer rat initially given eggs of H. nana became completely immune to egg challenge within 2 days of egg inoculation; no cysts derived from challenge eggs were found in the immunized rat. Immunosuppression, assessed by the success of cyst recovery in the tissue 4 days after egg challenge, had no promotive effect on the recovery of adult worms derived from eggs initially inoculated. Rats initially given eggs and immunosuppressed by cyclophosphamide or antithymocyte serum did not harbor any adult worms. Cortisone acetate treatment which was sufficient for eggs inoculated to mature (a total of 75 or even 200 mg, from Day 5 of egg inoculation) had no effects of immunosuppression, whereas cortisone acetate treatment which was sufficient for immunosuppression (a total of 150 mg from Day -2, two days prior to the initial egg inoculation) induced some adult formation as well. In addition, when mouse-derived cysts were inoculated into the rat instead of eggs, they also failed completely to mature even when the rat was treated with cyclophosphamide or antithimocyte serum. However, when the rat was treated with cortisone acetate from the day of cyst inoculation, the cysts developed into adult worms. Therefore, these results indicate that the Fischer rat clearly differs in its susceptibility to the tissue phase of egg inoculation and to the lumen phase of cyst inoculation of H. nana, and strongly suggest that the failure of maturation of H. nana in the unnatural host Fischer rat is not attributed to innate and/ or acquired immunity of the rat but to other nonimmunological mechanisms.     

Electron microscope studies of Fasciola hepatica. X. Egg formation

Evidence obtained would suggest that the processes involved in egg formation in Fasciola hepatica occur in the following sequence. The egg constituents, namely an ovum and some vitelline cells, pass through the proximal ootype and as they do so they are smeared by the secretions of Mehlis' gland which have accumulated there. A temporary interface is set up between the Mehlis' gland secretion and the fluid which surrounds the egg constituents. Shell globules are released by the vitelline cells and coalesce on the inner aspect of the interface. At the same time some of the Mehlis' secretion diffuses across the interface, thereby bringing about the dissociation of the interface. It is suggested that Mehlis' gland secretion brings about the fusion of the shell layer. During the initial stages of the process the developing egg is supported by the cells of the ootype epithelium. Later the egg passes into a wider, more distal part of the ootype where the process of shell deposition is continued. When complete, or almost complete, the egg passes into the uterus where changes indiciating the initial process of tanning are seen to take place. A thin, very uniform layer of Mehlis' gland secretion can still be identified on the surface of mature eggs.     

Low transformation growth factor-β1 production and collagen synthesis correlate with the lack of hepatic periportal fibrosis development in undernourished mice infected with Schistosoma mansoni

Undernourished mice infected (UI) submitted to low and long-lasting infections by Schistosoma mansoni are unable to develop the hepatic periportal fibrosis that is equivalent to Symmers’ fibrosis in humans. In this report, the effects of the host’s nutritional status on parasite (worm load, egg viability and maturation) and host (growth curves, biology, collagen synthesis and characteristics of the immunological response) were studied and these are considered as interdependent factors influencing the amount and distribution of fibrous tissue in hepatic periovular granulomas and portal spaces. The nutritional status of the host influenced the low body weight and low parasite burden detected in UI mice as well as the number, viability and maturation of released eggs. The reduced oviposition and increased number of degenerated or dead eggs were associated with low protein synthesis detected in deficient hosts, which likely induced the observed decrease in transformation growth factor (TGF)-β1 and liver collagen. Despite the reduced number of mature eggs in UI mice, the activation of TGF-β1 and hepatic stellate cells occurred regardless of the unviability of most miracidia, due to stimulation by fibrogenic proteins and eggshell glycoproteins. However, changes in the repair mechanisms influenced by the nutritional status in deficient animals may account for the decreased liver collagen detected in the present study.     

Parasitological and morphological study of Schistosoma mansoni and diabetes mellitus in mice

Schistosomes are blood-dwelling flukes which are highly dependent on the host metabolism. The aim of this study was to investigate possible relationship between streptozotocin-induced diabetes and the outcome of acute murine schistosomiasis mansoni. Male and female SW mice were treated by a single intraperitoneally injected dose of streptozotocin (180 mg/kg). Seven days after induction, both control and diabetic animals were infected with 70 Schistosoma mansoni cercariae (BH strain). Diabetics and their controls were weighed 45 days after birth and for the last time prior to killing. Susceptibility to infection was evaluated twice a week by quantifying fecal egg excretion 7–9 weeks post-infection by the Kato–Katz’ thick smear method. Mice were euthanized the day after the last fecal examination was performed. Adult worms were recovered from the portal system and mesenteric veins, whereas liver and intestine were removed for enumeration of egg load. No differences in worm length or in measurements of the reproductive organs, tegument, and suckers were detected. Also oviposition was unaffected as the total number of eggs per female worm from the liver, the small and the large intestine was the same in both groups. An oogram evaluation revealed a lower percentage of mature (23.0% vs. 40.7%) and a higher percentage of immature (69.1% vs. 51.7%) eggs in the small intestine of the diabetic mice. We suggest that principally a hampered egg passage through the intestine tissue caused this reduction and that probably both the eggs and the impaired host response play a role.     

Human Infections with Liver and Minute Intestinal Flukes in Guangxi, China: Analysis by DNA Sequencing, Ultrasonography, and Immunoaffinity Chromatography

The prevalence of liver and intestinal fluke infections was determined by surveying inhabitants of Hengxuan, Fusui, and Shanglin villages which were known to be endemic for liver flukes in Guangxi, China in May 2010. A total of 718 people were examined for helminth eggs by the Kato-Katz thick smear technique, ultrasonography, immunoaffinity chromatography, and DNA sequencing. The overall egg positive rate was found to be 59.6% (28.0-70.6%) that included mixed infections with liver and intestinal flukes. Cases showing higher than 20,000 eggs per gram of feces (EPG) were detected between 1.3% and 16.2%. Ultrasonographic findings exhibited overall 28.2% (72 of 255 cases) dilatation rate of the intrahepatic bile duct. Clonorchis sinensis infection was detected serologically in 88.3% (38 of 43 cases) among C. sinensis egg positive subjects by the immunoaffinity chromatography using a specific antigen for C. sinensis. For differential diagnosis of the liver and intestinal flukes, more precise PCR and nucleotide sequencing for copro-DNA were performed for 46 egg positive cases. Mixed infections with C. sinensis and Metagonimus yokogawai were detected in 8 of 46 egg positive cases, whereas 29 specimens were positive for Haplorchis taichui. Ultrasonographic findings and immunoaffinity chromatography results showed usefulness, even in a limited way, in figuring out of the liver fluke endemicity.     

Fasciola hepatica in Snails Collected from Water-Dropwort Fields using PCR

Fasciola hepatica is a trematode that causes zoonosis mainly in cattle and sheep and occasionally in humans. Fascioliasis has been reported in Korea; however, determining F. hepatica infection in snails has not been done recently. Thus, using PCR, we evaluated the prevalence of F. hepatica infection in snails at 4 large water-dropwort fields. Among 349 examined snails, F. hepatica-specific internal transcribed space 1 (ITS-1) and/or ITS-2 markers were detected in 12 snails and confirmed using sequence analysis. Morphologically, 213 of 349 collected snails were dextral shelled, which is the same aperture as the lymnaeid snail, the vectorial host for F. hepatica. Among the 12 F. hepatica-infected snails, 6 were known first intermediate hosts in Korea (Lymnaea viridis and L. ollula) and the remaining 6 (Lymnaea sp.) were potentially a new first intermediate host in Korea. It has been shown that the overall prevalence of the snails contaminated with F. hepatica in water-dropwort fields was 3.4%; however, the prevalence varied among the fields. This is the first study to estimate the prevalence of F. hepatica infection using the vectorial capacity of the snails in Korea.     

Continuous Variation Rather than Specialization in the Egg Phenotypes of Cuckoos (Cuculus canorus) Parasitizing Two Sympatric Reed Warbler Species

The evolution of brood parasitism has long attracted considerable attention among behavioural ecologists, especially in the common cuckoo system. Common cuckoos (Cuculus canorus) are obligatory brood parasites, laying eggs in nests of passerines and specializing on specific host species. Specialized races of cuckoos are genetically distinct. Often in a given area, cuckoos encounter multiple hosts showing substantial variation in egg morphology. Exploiting different hosts should lead to egg-phenotype specialization in cuckoos to match egg phenotypes of the hosts. Here we test this assumption using a wild population of two sympatrically occurring host species: the great reed warbler (Acrocephalus arundinaceus) and reed warbler (A. scirpaceus). Using colour spectrophotometry, egg shell dynamometry and egg size measurements, we studied egg morphologies of cuckoos parasitizing these two hosts. In spite of observing clear differences between host egg phenotypes, we found no clear differences in cuckoo egg morphologies. Interestingly, although chromatically cuckoo eggs were more similar to reed warbler eggs, after taking into account achromatic differences, cuckoo eggs seemed to be equally similar to both host species. We hypothesize that such pattern may represent an initial stage of an averaging strategy of cuckoos, that – instead of specializing for specific hosts or exploiting only one host – adapt to multiple hosts.     

Draft Genome Sequences of 21 Salmonella enterica Serovar Enteritidis Strains

Salmonella enterica subsp. enterica serovar Enteritidis is a common food-borne pathogen, often associated with shell eggs and poultry. Here, we report draft genomes of 21 S. Enteritidis strains associated with or related to the U.S.-wide 2010 shell egg recall. Eleven of these genomes were from environmental isolates associated with the egg outbreak, and 10 were reference isolates from previous years, unrelated to the outbreak. The whole-genome sequence data for these 21 human pathogen strains are being released in conjunction with the newly formed 100K Genome Project.     

Trichogramma chilonis Ishii: A potential biological control agent of Crocidolomia pavonana in Samoa

Crocidolomia pavonana F. (Lepidoptera: Crambidae) is a major pest of Brassica crops in tropical and sub-tropical regions of Africa, Asia and the Pacific. There are no previous reports of effective natural enemies of the pest across this range but in Samoa an arrhenotokous population of the generalist egg parasitoid Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae) frequently attacks it. This is the first record of T. chilonis in Samoa. A three-year field recruitment study showed that although C. pavonana eggs occurred at all times of the year, their abundance was greatest during drier periods. Parasitism of C. pavonana egg masses by T. chilonis was variable (0–87% of egg masses attacked) but the parasitoid was recovered from eggs collected at all times of the year and it is well established in the major Brassica growing regions of the island of Upolu. When partial lifetables were constructed for C. pavonana, the rate of egg disappearance (likely due to predation and the physical effects of rainfall) ranged from 0 to 0.839 and the marginal rate of mortality due to T. chilonis ranged from 0 to 0.474. When it was present, T. chilonis was the major mortality factor affecting C. pavonana eggs in all but one of the recruitment studies. The historical problems surrounding the identity and species status of T. chilonis are discussed and its host range and distribution in the Asia-Pacific region is reviewed briefly. Finally, the potential of this population of T. chilonis for development as a biological control agent of C. pavonana is considered.     

Recovery and Longevity of Egg Masses of Meloidogyne incognita during Simulated Winter Survival

Effects of soil matrix potential on longevity of egg masses of Meloidogyne incognita were determined during simulated winter conditions. Egg masses were recovered from isolated root fragments incubated in field soil at matrix potentials of -0.1, -0.3, -1.0, and -4.0 bars throughout winter survival periods of 10 weeks for tomato roots and 12 weeks for cotton roots. Egg masses were more superficial on cotton roots than on tomato roots and were more easily dislodged from cotton roots during recovery of root fragments by elutriation. The rate of decline in numbers of eggs and J2 per egg mass was greater in wet as compared to dry soils (P = 0.01), with the relationship between numbers of eggs and J2 per egg mass and time being best described by quadratic models. Percentage hatch of recovered eggs declines linearly with time at soil matrix potentials of -0.1 and -0.3 bars, but at -1.0 and -4.0 bars the percentage hatch of recovered eggs increased before declining. Effects of soil matrix potential on numbers of eggs per egg mass and percentage hatch of recovered eggs were consistent with previous reports that low soil moisture inhibits egg hatch before affecting egg development. Estimations of egg population densities during winter survival periods will be affected by ability to recover infected root fragments from the soil without dislodging associated egg masses. There is a need for procedures for extraction of egg masses not attached to roots from the soil.     

Influence of Temperature on Multiplication and Egg Hatching of Longidorus africanus

Longidorus africanus multiplication on tomato was highest at 29 °C. Few nematodes were recovered after 6 weeks at soil temperatures of 35 °C or below 23 °C. The time to egg hatching was shortest and the percentage of eggs hatching was highest at 29 °C. The minimum temperature and the heat sum above this temperature required for egg development were calculated to be 14.3 °C and 94.08 degree-days, respectively. The thermal times required for egg development by L. africanus and L. elongatus were nearly identical. For both species the product of the base temperature and the heat sum was near constant, and at a temperature of 22.3 °C the rates of egg development were equal.     

Plant-Induced Hatching of Eggs of the Soybean Cyst Nematode Heterodera glycines

Root diffusate from soybean plants caused greater hatching of Heterodera glycines eggs during vegetative growth of the host, but the activity declined with plant senescence. Chelation of the root diffusate with ethylenediamine tetraacetic acid (EDTA) significantly increased hatching activity for H. glycines eggs. Diffusate from leafless plants caused little hatching, whereas treatment of intact plants with the growth regulators gibberellin and kinetin had no effect on the hatching activity of root diffusate. Treating H. glycines eggs with zinc chloride and root diffusate reduced egg hatching from zinc chloride alone. Levels of zinc in the root diffusate were insufficient to induce egg hatch, based on analysis by atomic absorption spectrophotometry. The enzymatic activity of leucine aminopeptidase in H. glycines eggs was not altered by treatment with chelated or nonchelated root diffusate.