Surface and internal tegumental changes in juvenile Fasciola hepatica following treatment in vivo with the experimental fasciolicide, compound alpha

Eight indoor-reared, crossbred sheep with no pre-exposure to Fasciola hepatica were infected, by oral gavage, with 200 metacercarial cysts of the triclabendazole-susceptible, Cullompton isolate of F. hepatica. Anthelmintic dosing occurred at 4 weeks post-infection using 15mg/kg compound alpha. Two treated sheep per time period were euthanized at 24h, 48h and 72h post-treatment with compound alpha. The two sheep from the control group were euthanized alongside the 24h alpha-treated sheep. Juvenile flukes were recovered from each of the sheeps' liver and processed for examination by electron microscopy. The surface morphology of the flukes' tegument was assessed using scanning electron microscopy (SEM). The ultrastructure of the tegumental syncytium and underlying tegumental cells and connections and somatic musculature were investigated using transmission electron microscopy (TEM). Both the SEM and TEM results revealed a level of disruption that increased with time, culminating at 72h with extensive tegumental loss and substantial degeneration of the cell bodies. The effects of compound alpha on the surface morphology were not particularly apparent until 48h post-treatment, when disruption included swelling and blebbing of the tegument. At 72h post-treatment, SEM revealed loss of the entire syncytial layer over large areas of the flukes. In the areas where the syncytium was lost and the basal lamina exposed, lesions of varying sizes had developed, revealing underlying tissues. Though minor forms of disruption to the ultrastructure of the syncytium were observed using TEM 24h post-treatment, it was at 48h post-treatment that substantial stress responses occurred. They included the presence of autophagic vacuoles and 'open' bodies at the apex of the syncytium and swelling of the basal infolds. The mitochondria within the syncytium and tegumental cells became progressively more disrupted over the three time periods and, by 72h post-treatment, they were frequently distorted and swollen in appearance, and contained severely swollen cristae. By 72h, the number of secretory bodies, particularly T1 bodies, had become significantly depleted in their respective cell bodies, cytoplasmic processes and in the tegumental syncytium. Both the circular and longitudinal muscle bundles were severely disrupted 72h post-treatment. They frequently contained a reduced number of muscle fibres and, in more severe instances, there was an absence of fibres altogether.     

An in vitro effect of propolis on adult worms of Fasciola gigantica

The effect of Siwa propolis on adult flukes was evaluated using scanning electron microcopy. It gave an overview of the surface architecture of the tegument of Fasciola gigantica apical cone. The base of the spines appeared to be "flaking off" and showed severe blebbing after 24h incubation with 10 micro/ml propolis. This swelling became so sever and the spines were barely visible, on increasing the concentration to 20 micro/ml. Besides, there were many large blebs on the apical cone, a number of which appeared to have burst, causing lesions and the tegument was marked by a number of pits caused by the loss of spines. With the higher concentration of 30 micro/ml, erosion of the surface had occurred to such extent that no tegument remained, only a mass of fibrous structures. The tegumental changes occurred following incubation in propolis were compared with those observed with triclabendazole (TCBZ) "Fasinex" because of its high efficacy against both mature and immature flukes.     

Time-dependent changes to the tegumental system and gastrodermis of adult Fasciola hepatica following treatment in vivo with triclabendazole in the sheep host

Eight indoor-reared cross-bred sheep with no pre-exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. At 12 weeks post-infection, sheep were dosed with 10mg/kg triclabendazole. Two sheep per time period were euthanized at 48 h, 72 h and 96 h post-treatment (pt). Two control sheep were euthanized alongside the 96 h triclabendazole-treated sheep. Flukes were recovered from each of the sheeps liver and, if present, from the gall bladder and they were processed for transmission electron microscopy (TEM). Disruption to the ultrastructure of the tegument became increasingly severe over time pt. Flukes recovered at 48 h pt showed widespread blebbing of the apical plasma membrane and swelling of the mucopolysaccharide masses surrounding the basal infolds. There was evidence of reduced secretory activity in the tegumental cells and spacing between the cells. Sloughing of the tegumental syncytium was observed at 72 h pt. The subtegumental musculature, parenchyma and tegumental cells were severely disrupted. At 96 h pt, all of the flukes were totally devoid of tegument. Disruption to the subtegumental tissue and somatic musculature was severe, and was so extreme in some specimens that the tegumental cells were barely discernible. Disruption to the gastrodermis was also progressive, though not as severe as disruption to the tegument. There was a general decline of secretory activity with time pt. Autophagic activity was apparent from 48 h pt and became more widespread with increasing time, culminating in breakdown of the gastrodermal cell cytoplasm. The mitochondria were swollen and electron-lucent and the cisternae of the granular endoplasmic reticulum were dilated and fragmented from 72 h pt.     

Actin in Stratified Squamous Keratinized Epithelium*

Actin filament distribution patterns were revealed in a stratified squamous keratinized epithelium using phalloidin-fluorescent and immunogold labeling techniques applied on bovine luminal pilar as a model tissue. In non-keratinized cell types, actin concentrates on the microfilament-rich cellular cortex as well as on cytoplasmic processes and protrusions. In cornified cells labeling is distributed diffusely over the amorphous cytoplasm. A constant feature in all cell types is plasmalem-mal labeling. Desmosomes exhibit deposition on their plasmalemmal leaflets, the dense central stratum and plaques. Desmosomal as well as cytoplasmic keratinfilament bundles also label for actin, the latter often in a cross-banded manner. These cellular distribution patterns of actin filaments are discussed with respect to the singificance of the microfilaments in the process of cell shape determination, stratification, and cell adhesion.     

Cytoskeletal changes in <Emphasis Type="Italic">Eimeria bovis</Emphasis>-infected host endothelial cells during first merogony

The first merogony of Eimeria bovis takes place in lymphatic endothelial cells of the ileum, resulting in the formation of macromeronts up to 250 mum. In this study, we investigated the host cell cytoskeleton (actin filaments, microtubules, spectrin, vimentin intermediate filaments) associated with parasitic development in vitro by confocal laser scanning microscopy (CLSM) using primary bovine umbilical vein endothelial cells (BUVEC) and bovine spleen lymphatic endothelial cells (BSLEC) as host cells. No prominent changes in the host cell cytoskeleton were detected 1-3 days after E. bovis sporozoite invasion. With ongoing meront maturation a significant increase in microtubules and actin filaments close to the parasitophorous vacuole (PV) was found. Mature macromeronts within the PV were completely enclosed by these cytoskeletal elements. Our findings suggest, that in order to guarantee the survival of the host cell on the enlargement of macromeronts, E. bovis needs not only to augment but also to rearrange its cytoskeletal system.     

Comparative study of the reproductive organs of Fasciola groups by optical microscope

Reproductive organs of stained and mounted whole specimens of different types of Fasciola (F. hepatica, F. gigantica, and parthenogenetic diploid and triploid flukes) were observed to clarify the structure of their reproductive organs. The results are as follows; 1. Basic structure differences could not be identified. 2. The flukes without sperm, or those with an extremely small quantity in the seminal vesicle. are parthenogenetic Fasciola sp. 3. It was newly discovered that the surface of the cirrus is surrounded by many shallow gutters, and that spines form a line in the gutters. 4. The structure of the reproductive organ on the genus Fasciola are shown in detail in the figures.     

Myosins 1 and 6, myosin light chain kinase,actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes

Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.     

Intraocular and orbital malignant schwannomas in F344 rats.

Intraocular and orbital malignant Schwannomas in two F344 rats are presented. The two Schwannomas were identified among approximately 60,000 male and 60,000 female F344 rats. The intraocular malignant Schwannoma occurred in the iris, invading the corneal stroma through the destroyed Descemet membrane. The malignant orbital Schwannoma occurred in the left orbit, invading the contralateral orbit along the optic nerve. Histologically, the intraocular Schwannoma consisted predominantly of a perivascular fascicular pattern of plump spindle cells associated with marked cytoplasmic vacuolization. The orbital Schwannoma consisted of Antoni type A and B pattern, but Antoni B tissues predominated. Antoni A tissues consisted of closely packed, elongated spindle cells arranged in interlacing fascicles, while Antoni B tissues were highly cellular and consisted of anaplastic, small cells associated with marked cyst formation. Immunohistochemically, the intraocular Schwannoma had a positive immunoreactivity for S-100 protein, while the orbital Schwannoma had a negative immunoreactivity. Ultrastructurally, the cells of both intraocular and orbital Schwannomas had long, thin cell processes and pericytoplasmic basal laminae. Particularly, the plump spindle cells of the intraocular Schwannoma were most strikingly characterized by the well developed, extremely attenuated cell processes arranged in a lamellar or spiral pattern. These cell processes and cell bodies were associated with numerous desmosomes. Intracytoplasmic filamentous granules and bodies, consisting of intermediate filaments approximately 7 nm in width, were additional characteristics of the plump spindle cells.     

Bovine T cell responses to recombinant thioredoxin of Fasciola hepatica

Fasciolosis is an economically significant disease of ruminants, caused by infection with the digenetic trematodes, Fasciola hepatica and F. gigantica. Some vaccination trials using irradiated metacercariae or isolated proteins have been shown to afford significant protection. However, the mechanisms of specific immunity against this pathogen have not been elucidated. We have identified thioredoxin, a tegument antigen of F. hepatica, among several proteins that are common to both the juvenile and adult fluke within the mammalian host and have undertaken studies to characterize bovine T cell responses to recombinant thioredoxin protein (FH 2020). Peripheral blood mononuclear cells from immune cattle proliferated specifically to crude F. hepatica antigenic extract but not to FH 2020. However, after repeated stimulation of lymphocytes by alternating crude extract and FH 2020, FH 2020-specific proliferation by T cell lines was observed. T cell clones were subsequently generated and found to respond specifically but weakly to both crude antigen and FH 2020. Thioredoxin appears to be only weakly antigenic for bovine T cells and is, therefore, an unpromising candidate for inducing resistance to F. hepatica.     

Cellular and humoral responses in liver of cattle and buffaloes infected with a single dose of Fasciola gigantica

The cellular components of the hepatic inflammatory infiltrate in cattle and buffaloes infected with a single dose of 1000 Fasciola gigantica were analysed by immunohistochemistry and histology. T and B lymphocytes, plasma cells, eosinophils and mast cells were present in the hepatic lesions. It is proposed that both cellular and humoral immune responses were induced in the liver of cattle and buffaloes during infection with F. gigantica probably by antigens released by the developing flukes and by damage caused by the flukes during their migration in the liver. The local T cell response differed between these animals, with the response decreasing after 3 weeks post-infection in cattle in contrast to a gradually increasing response in buffaloes. Difference in the T cell response between cattle and buffaloes may be related to their differences in resistance and resilience to infection with F. gigantica.     

Methods for in vitro study of the invasive processes of Fasciola gigantica.

In vitro techniques have been developed to study the invasive processes of Fasciola gigantica. The conditions necessary for excystment were identical with those rquired by F hepatica. The ability of larvae to penetrate mouse gut in vitro was influenced by the composition of the fluid within the gut, and by the region of gut wall to which the larvae were exposed. Larvae remained viable on spleen cell monolayers for at least 60 days.     

The tegument of Schistosoma hippopotami from Hippopotamus amphibius in the Kruger National Park

Schistosoma hippopotami were collected from the right heart chambers and pulmonary arteries of Hippopotamus amphibius culled in the Kruger National Park. The schistosomes were subjected to scanning electron microscopy as well as optical microscopy. The results indicate that S. hippopotami is not conspecific to S. mansoni as suggested in the literature. On account of the morphology of certain tegumental structures of both male and female parasites, it is suggested that S. hippopotami is adapted to the pulmonary arterial circulation of its host.     

Enhancement of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica by co-treatment with ketoconazole

An in vivo study in the laboratory rat model was carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from combination treatment of triclabendazole (TCBZ) and the metabolic inhibitor, ketoconazole (KTZ). Rats were infected with the TCBZ-resistant Oberon isolate of F. hepatica and divided into 3 groups at 12 weeks post-infection. The first group was dosed orally with TCBZ at a dosage of 10mg/kg and KTZ at a dosage of 10mg/kg. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.). A second group of rats was treated with TCBZ alone (10mg/kg) and sacrificed at 96 h p.t. The third group acted as untreated controls. Surface changes were monitored by scanning electron microscopy (SEM). In flukes from the TCBZ+KTZ-treated group, the results showed a progressive and time-dependent increase in the level of disruption to the tegumental syncytium. Swelling, furrowing, blebbing and sloughing of the syncytium increased with time p.t. Another feature seen was a thick layer of tegumental shedding in some fluke samples at different times p.t. By comparison, flukes treated with TCBZ alone remained unaffected. The results demonstrated that the Oberon isolate is only sensitive to drug action in the presence of ketoconazole, indicating that combining triclabendazole with a metabolic inhibitor could be used to preserve the effectiveness of the drug against TCBZ-resistant populations of F. hepatica.     

High resistance to experimental infection with Fasciola gigantica in Javanese thin-tailed sheep

Innate resistance of Javanese thin-tailed sheep to Fasciola gigantica was investigated in animals infected with single doses of 150 or 500 metacercariae and killed 4, 8, 12 or 16 weeks after infection. Infected and non-infected sheep had similar values for packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin concentration, serum glutamate dehydrogenase, serum gamma glutamyl transferase and serum aspartate transferase throughout the trial, except for one animal infected with 500 metacercariae from which the highest recovery of flukes (55) was made. This animal developed pathologically altered values from 12 weeks post infection, coincident with the period of greatest hepatic haemorrhage and destruction of hepatic tissue by migrating flukes and their entry into bile ducts. However, values were altered much less than those reported in other sheep given as few as 200 metacercaria of F. gigantica. Both susceptibility to infection with F. gigantica, as indicated by percentage take of metacercariae and the severity of pathological changes were low in this study in comparison with reports involving other breeds of sheep infected with this parasite. These findings support the conclusion that Javanese thin-tailed sheep have a high innate resistance to F. gigantica.     

Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection

This study reports the early biochemical changes in plasma, comparative host-immune responses and parasite recovery data in Merino sheep during the first 10 weeks of infection with Fasciola gigantica and Fasciola hepatica. One group of sheep were uninfected, four groups of sheep received incremental challenge doses of F. gigantica metacercariae (50, 125, 225 and 400, respectively) and the sixth group was challenged with 250 F. hepatica metacercariae. At 10 weeks post infection (wpi), sheep challenged with F. hepatica showed the greatest fluke recovery (mean 119, range 84-166); a significantly higher biomass of parasites recovered (2.5-fold greater than the highest dose of F. gigantica); and a greater mean % parasite recovery (39.3%, range 27-55%) than any group challenged with F. gigantica. Within the groups dosed with F. gigantica a strong dose-dependent response was observed in both fluke recovery and fluke biomass with increasing dose of metacercariae. The mean % parasite recovery of F. gigantica infected groups 1-5 were 26, 23, 26 and 25%, respectively, suggesting a uniform viability of parasite establishment independent of infection dose. At 6 wpi, elevated levels of plasma GLDH were observed in the F. gigantica infected groups compared to the uninfected sheep (p<0.005) whereas the F. hepatica challenged group had four-fold higher levels of GLDH compared to the F. gigantica infected group (p<0.001). Elevated levels of GGT as an indicator of epithelial damage in the bile duct was only seen in the group challenged with F. hepatica at 10 wpi when it rose from below 100 IU/l to approximately 250 IU/l (p<0.0001) whereas no detectable increase in GGT was observed in any of the groups challenged with F. gigantica. The white blood cell response to F. hepatica infection was biphasic with the initial peak at 4 wpi and a second peak at 9 wpi, corresponding to the period of migration of juvenile fluke in the liver and the time when adult flukes are migrating into the bile duct, respectively. This biphasic response was also evident in the changes in the eosinophil counts and serum haemoglobin levels. There was a trend toward higher parasite-specific IgG2 titres in sheep infected with lower worm burdens, suggesting that higher F. gigantica or F. hepatica burdens suppress IgG2 responses. The findings of this study suggest that, in early infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica because of its rapid increase in size and the speed of its progression through the migratory phases of its life cycle.     

两种片形吸虫感染家兔的试验比较

用大片形吸虫和肝片形吸虫感染家兔以便选择大片形吸虫对动物的最佳感染量,及明确肝片形吸虫和大片形吸虫的生物学和对动物宿主的致病力的差别。结果显示肝片形吸虫虫体在兔体内发育成熟的时间早于大片形吸虫,感染成活率更高,对动物的病理损害明显比感染大片形吸虫兔的病变要轻。本试验证实这两种片形吸虫除了形态学的差异外,在对动物致病力、病理损害等方面确实存在差别。     

The corticosteroid content of cattle skin washings.

In vitro techniques have been developed to study the invasive processes of Fasciola gigantica. The conditions necessary for excystment were identical with those required by F hepatica. The ability of larvae to penetrate mouse gut in vitro was influenced by the composition of the fluid within the gut, and by the region of gut wall to which the larvae were exposed. Larvae remained viable on spleen cell monolayers for at least 60 days.     

The purification and characterization of a cysteine protease of Fasciola gigantica adult worms.

A 26-28 kDa protease was isolated from Fasciola gigantica adult worms by a two-stage purification process of column chromatography in a Sephacryl S-200 column and affinity chromatography in an L-phenylalanine-agarose column. This protease is a cysteine (thiol) proteinase with an optimum pH of 4.5 and is not inhibited by anti F. gigantica immunoglobulin G. The enzyme was inhibited by protease inhibitors known to inhibit cysteine proteases but not by metallo-, aspartate or serine protease inhibitors. The effect of several protease inhibitors and anti-F, gigantica IgG was also assessed on the total proteolytic activity of F. gigantica. There appears to be a preponderance of cysteine protease activity in F. gigantica and there was a significant inhibition of total proteolytic activity by anti-F. gigantica IgG.     

Electron microscopic studies of the morphogenesis of duck enteritis virus

The morphogenesis of duck enteritis virus (DEV) and distribution in vivo were observed by electron microscopy after ducks were infected experimentally with DEV virulent strain. The investigation showed that a few typical herpesvirus virions and nucleocapsids were first observed in the spleen, thymus, and bursa of Fabricius (BF), and many nucleocapsids, mature viruses, and viral inclusion bodies could be found in the nucleus and cytoplasm of infected liver, small intestine, spleen, thymus, and BF when the ducks died. Nucleocapsids assembled both in nucleus and cytoplasm and could be divided into four different types according to their structures. Typical herpesvirus, light particles (L-particles), and virions without tegument could be observed at the same time. With the replication, assembly, and maturation of the viruses, intracytoplasmic and intranuclear inclusion bodies, electron-density particles, microtubules, hollow tubes, and coated electron-density bodies were observed in infected cells.     

Subcutaneous Soft Tissue Sarcoma with Rhabdoid Features in a Dog

A nine-year-old male beagle dog had a white spherical mass in the subcutis of the left lumbar region. Microscopically, spindle to oval cells diffusely proliferated in the fibrous and myxoid stroma. Many neoplastic cells showed rhabdoid features or vacuolated cytoplasm. Immunohistochemically, the neoplastic cells were positive for vimentin and S100 and partly positive for neuron-specific enolase and glial fibrillary acidic protein but were negative for von Willebrand factor, desmin and α-smooth muscle actin. Ultrastructurally, the neoplastic cells had abundant cytoplasmic processes and desmosome-like structures. Cytoplasmic inclusions of rhabdoid-featured cells in HE sections were composed of aggregates of intermediate filaments, and cytoplasmic vacuoles were identified as an invagination of cytoplasm. Although malignant peripheral nerve sheath tumor was suggested according to these results, the present case was diagnosed as a soft tissue sarcoma with rhabdoid features due to a lack of identification of the basal lamina under electron microscopy.