A preliminary study to understand the effect of Fasciola hepatica tegument on naïve macrophages and humoral responses in an ovine model

Fasciola hepatica, the liver fluke, is a highly evolved endo-parasite that uses various mechanisms to evade the host immune system. The immunosuppressive capabilities of the parasite's excretory/secretory products have been well demonstrated by previous independent studies. However, the role of the parasite's tegument in the immune responses remains to be investigated. In this study, the effect of the tegument and other fractions of adult F. hepatica (excretory/secretory, liver fluke homogenate and liver fluke homogenate without tegument) in the activation of naïve macrophages in vitro was investigated using an ovine model. In addition, an immunoproteomic approach was used to investigate the characteristics of humoral antibody responses developed in sheep against the tegument fraction. The results indicated significantly increased arginase expression in macrophages incubated with the tegument and excretory/secretory fractions. Two dimensional gel electrophoresis of the tegument demonstrated approximately 100 protein spots, with only four of these spots were highly reactive with the positive serum as determined by 2-DE immunoblotting. These results give a preliminary indication that the liver fluke tegument may play role in avoiding hosts’ protective immune responses against itself.     

Tegumental surface changes in juvenile Fasciola hepatica in response to treatment in vivo with triclabendazole

Eight indoor-reared crossbred 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. Anthelmintic dosing occurred at 4 weeks post-infection with 10 mg/kg triclabendazole. Two treated sheep were euthanized at 48 h, 72 h and 96 h post-treatment with triclabendazole. Two control sheep were euthanized alongside the 48 h triclabendazole-treated sheep. Juvenile flukes were recovered from each of the sheeps' liver and processed for scanning electron microscopy (SEM). Flukes were still active 48 h post-treatment and displayed limited morphological disruption. There was some blebbing and sloughing of the tegument around the oral sucker. In several of the specimens, an extra layer had been deposited on the fluke surface, giving it a flattened appearance. At 72 h post-treatment, only one fluke remained alive and the disruption varied in degree. In the majority of flukes, there was severe swelling of the tegument, accompanied by isolated areas of flattening along the lateral margins of the flukes and in the tail region. Limited areas of sloughing occurred in the tail region. In more seriously affected specimens, the syncytium had been stripped away to reveal the basal lamina and some deeper lesions were also observed. By 96 h post-treatment, all the flukes were dead and were grossly disrupted. They were totally devoid of tegument and deep lesions exposed the internal tissues of the fluke.     

Site adaptations of Acanthogyrus (Acanthosentis) tilapiae: Observations through light and scanning electron microscopy

Acanthogyrus (Acanthosentis) tilapiae parasites were collected from the intestines of 300 fish belonging to three tilapia species sourced at the River Nile, Giza, Egypt. The proboscis of the parasite was characterized by three rows of hooks that curved towards the posterior of the body. The first row is supported by unmodified hooks. The parasite tegument has a series of alternative folds and a large number of pores. Sensory ganglia are located on the surface of the proboscis and body. Acanthogyrus (Acanthosentis) tilapiae provokes an aggressive host response indicated by hyperplasia of the intestinal goblet cells and focal eosinophil infiltrations. This acanthocephalan parasite shows a highly modified adaptation to its site of host infection.     

台湾次睾吸虫成虫体被的扫描电镜观察

台湾次睾吸虫１５日龄成虫经扫描电观察，虫体整个背面和腹面前４／５的体表皮层着生有鱼鳞状的簇生体棘，簇生体棘在虫体前端分布较密集，由前向后渐变稀疏，每一个簇生体棘中含３－６个尖刀形棘。口盘上有１５－１８个无纤毛圆丘型乳突，腹吸盘唇部光滑无乳突分布，但在其周围可见到１０余个花蕾状乳突。     

Characteristics of mature oocytes from four species of marine teleosts

The morphology and histology of the digestive tract of the branchiuran crustacean, Chonopeltis australis Box shall, 1976 are described from serial sections. The foregut is differentiated into a preoral cavity, containing the mandibles and tongue, an ascending oesophagus, with an H-shaped lumen invested with longitudinal, circular and dilator muscles, a horizontal oesophagus with a star-shaped lumen and lacking longitudinal and dilator muscles, and an oesophageal funnel consisting of inner and recurrent walls. The midgut is differentiated into anterior and posterior chambers, separated by an S-shaped muscular tube. The arborescent midgut glands open laterally into the anterior part of the anterior midgut. Columnar epithelial cells line the anterior midgut whereas tall, papilliform cells are present in the epithelium of the posterior midgut. The transition from posterior midgut to hindgut is marked by the presence of very tall epithelial cells. The terminology describing the various parts of the digestive tract of branchiurans is discussed.     

Biochemical Aspects of Schistosome Behaviour

The haematophagous blood tiematodes that comprise the genus Schistosoma live in a stable environment with an adequate food supply and an efficient waste disposal system.

The main food is glucose obtained from the host blood and assimilated through the tegument Other dietary factors are assimilated through the tegument or the gut epithelium. The male fluke supplements and augments the diet of the female who is thus able to concentrate on egg production.

Live flukes resist attack by the host by secreting an immunologically inert envelope and by incorporating unchanged host serum protein into their structures. They can also incorporate host structural fatty acids unchanged.     

Potentiation of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica following its co-administration with the metabolic inhibitor, ketoconazole

An in vivo study in the laboratory rat model has been carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from co-treatment with triclabendazole (TCBZ) and ketoconazole (KTZ), a cytochrome P450 inhibitor. Rats were infected with the triclabendazole-resistant Oberon isolate of F. hepatica, dosed orally with triclabendazole at a dosage of 10 mg/kg live weight and ketoconazole at a dosage of 10 mg/kg live weight. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.) and changes to fluke ultrastructure were assessed using transmission electron microscopy (TEM). Results showed an increase in the severity of changes to the fluke ultrastructure with time p.t. Swelling of the basal infolds and the associated mucopolysaccharide masses became more severe with time. Golgi complexes, if present, were greatly reduced in size and number by 96 h p.t., and sub-tegumental flooding was seen from the 72 h time-period onwards. Some sloughing of the tegumental covering over the spines was observed at 96 h p.t. The results demonstrated that the Oberon isolate is more sensitive to TCBZ action in the presence of KTZ than to TCBZ alone, reinforcing the idea that altered drug metabolism is involved in the resistance mechanism. Moreover, they support the concept that TCBZ + inhibitor combinations (aimed at altering drug pharmacokinetics and potentiating the action of TCBZ) could be used in the treatment of TCBZ-R populations of F. hepatica.     

Anatomical and histological structure of the tongue and histochemical characteristics of the lingual salivary glands in the Chukar partridge (Alectoris chukar,Gray 1830)

1. The aim of the study was to examine the morphology of the tongue and the histochemical features of the lingual salivary glands in this species.

2. The tongue was elongated, terminating in a rather sharp, dagger-like apex. On the surface of the tongue and situated between the body and root of the tongue, two rows of conical papillae, the sharp apices of which pointed towards the posterior part of the tongue, were observed. The keratinised epithelium lining the dorsal surface lacked typical gustatory papillae. However, it was observed that taste buds were present in the epithelium of the lingual body and root. The tongue was supported by a structure composed of hyaline cartilage, the paraglossum, which extended from the lingual root to the apex. Simple branched tubular glands, which were encapsulated by connective tissue, were embedded within the submucosa in the body (anterior salivary glands) and root (posterior salivary glands) of the tongue. It was observed that the secretion of the lingual glands contained neutral mucins, proteoglycans containing carboxylic acid, weak and strong sulphated groups, N-acetylated sialomucins, but lacked glycogen.

3. It was demonstrated that, the general morphological features, papillary distribution of the tongue and the histological structure of the mucosa epithelium and the supportive elements displayed similarity to those of other domestic avian species. It was also determined that, in view of the particular feeding types, in the partridge, the presence of the papillary crest was not correlated with diet.     

Phase I biotransformation of albendazole in lancet fluke (Dicrocoelium dendriticum)

Dicroceliosis, a lancet fluke infection, is a frequent parasitosis of small ruminants and the anthelmintic drug albendazole (ABZ) is effective in control of this parasitosis. The aim of our project was to study the metabolism of ABZ and ABZ sulphoxide (ABZ.SO) in lancet fluke. Both invitro (subcellular fractions of fluke homogenates) and exvivo experiments (adult flukes cultivated in medium) were performed for this purpose. ABZ was metabolised invitro by lancet fluke NADPH-dependent enzymes by two oxidative steps (sulphoxidation and sulphonation). The apparent kinetic parameters of these reactions have been determined. In the exvivo experiments, only ABZ sulphoxidation was observed. The stereospecificity in ABZ sulphoxidation invitro was slight, with preferential formation of (+)-ABZ.SO enantiomer. In contrast (−)-ABZ.SO formation predominated in exvivo experiments. Sulphoreduction of ABZ.SO occurred neither invivo nor exvivo. The detection of ABZ oxidative metabolites indicates the presence of drug metabolising oxidases in lancet fluke.     

The life-cycle and seasonal abundance of Echinoparyphium montgomeriana n. sp. (Trematoda: Echinostomatidae) in Natal,South Africa

A new species of Echinoparyphium, E. montgomeriana, is described from Durban, South Africa. Certain unusual aspects of its morphology are discussed. The adult fluke is a parasite of the alimentary tract of birds. Its larval stages are common parasites of freshwater snails belonging to the family Planor-bidae including Buiinus africanus,the intermediate host ofSchistosoma haematobium and S. mattheei in the area. These intra-molluscan stages are shown to have a seasonal transmission cycle.     

Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Whole-Body Assay and Immunohistochemistry

Abstract

The sites of replication of infectious hematopoietic necrosis virus (IHNV) in infected tissues were detected in fingerling rainbow trout Oncorhynchus mykiss by in situ histologic techniques following immersion infection. Virus antigens in tissues were detected by a neutralizing mouse monoclonal antibody and a one-step anti-mouse biotin-streptavidin conjugated to horseradish peroxidase. The efficiency of infection and virulence of the virus determined by mortality rates showed high virulence of the selected IHNV isolates, and viral replication in individual fish showed that virus content of the fish increased rapidly from the second day to the seventh day postinfection. The earliest viral lesions following infection were detected in the epidermis of the pectoral fins, opercula, and ventral surface of the body. Virus lesions became evident in kidneys on the third day. By the fifth day, when there was a significant increase in virus titer, foci of viral replication were detected in gill tissue and in the anterior internal tissues below the epidermis. Subsequently, extensive virus replication and tissue destruction were observed in the spleen, dorsal adipose tissues, ventricle, and pseudobranch. Replication in the liver, the muscularis layers of the digestive tract, and the general body musculature followed later. These infection experiments indicated that the epidermis and gills of fish constitute important sites of early IHNV replication.     

Molecular and functional characterization of a Schistosoma bovis annexin: fibrinolytic and anticoagulant activity

Annexins belong to an evolutionarily conserved multigene family of proteins expressed throughout the animal and plant kingdoms. Although they are soluble cytosolic proteins that lack signal sequences, they have also been detected in extracellular fluids and have been associated with cell surface membranes, where they could be involved in anti-haemostatic and anti-inflammatory functions. Schistosome annexins have been identified on the parasite's tegument surface and excretory/secretory products, but their functions are still unknown. Here we report the cloning, sequencing, in silico analysis, and functional characterization of a Schistosoma bovis annexin. The predicted protein has typical annexin secondary and tertiary structures. Bioassays with the recombinant protein revealed that the protein is biologically active in vitro, showing fibrinolytic and anticoagulant properties. Finally, the expression of the native protein on the tegument surface of S. bovis schistosomula and adult worms is demonstrated, revealing the possibility of exposure to the host's immune system and thus offering a potential vaccine target for the control of schistosomiasis in ruminants.     

Scanning electron microscopy study of the lingual papillae in the European mole (Talpa europea, L., Talpidae)

The tongue in the adult European mole (Talpa europea L.) was examined by scanning electron microscope. The elongated tongue with a rounded apex is about 12-13 mm in length and 3-4 mm in width. On the apex the shallow median groove is present. On the dorsal surface of the lingual mucosa two types of mechanical papillae and two types of gustatory papillae were observed. Mechanical papillae are represented by numerous filiform papillae with a single process, covering the whole surface of the apex and body of the tongue, and massive conical papillae, found on the root of the tongue. The structure and density of filiform papillae varies in the anterior and posterior part of the tongue. A unique trait of the tongue in the European mole is the occurrence on the apex of the tongue of a single row of conical papillae. Gustatory papillae are represented by numerous fungiform papillae and one pair of vallate papillae. Dome-shaped fungiform papillae in the anterior part of the tongue are arranged linearly along both margins of the tongue, whereas in the posterior part of the body of the tongue flat fungiform papillae are distributed evenly among filiform papillae. Oval vallate papillae are surrounded by a continuous furrow and a single pad. In the posterior part of the root behind conical papillae the surface of the mucosa is flat with numerous orifices of lingual papillae located there. Observations on the distribution and structure of gustatory papillae in the common mole did not show the existence of special traits, differing them from those in terrestrial insectivores. The comparison of the morphology of the tongue, the distribution and structure of the lingual papillae in the European mole with those in the other species of Insectivores, indicated of a general similarity of features within the family Talpidae.     

Embryo and fetal position during pregnancy by ultrasonographic examinations in bottlenose dolphin (Tursiops truncatus)

From 2012 to 2017, serial ultrasonographic evaluation of 5 healthy bottlenose dolphins, Tursiops truncatus, were performed over the course of 6 pregnancies in Enoshima Aquarium. A total of 98 ultrasonographic examinations were included in the study. In three out of six cases, two embryos were observed between umbilicus and genital slit, and side of umbilicus in the dam’s body. All embryos were located in right below the peritoneum and observed from 308 to 325 days pre-partum. These days were corresponding to from 58 to 61 days after copulation respectively. The diameter of the embryo sac was approximately 4 cm. In three cases, the fetal head was located in the dam’s left lateral between umbilicus and genital slit from approximately 90 days pre-partum to the parturition. A snout of fetus is at the top of the uterine horn, and the tail lies close to the cervix. All six calves were fluke-first births (breech presentation). It was conjectured that the contraction of the dam’s uterus during parturition forced the fetus to invert, and the fetal tail fluke was expelled from the dam’s body. In three cases, judging from orientation of fetal tail fluke faced towards left side of the dam, a fetal position might be RSL (Right Sacrum-Lateral) within the birth canal. In the other three cases, the left and right positions of the fetus and the dam’s body are reversed during pregnancy and parturition.     

Morphometry on lancet flukes found in Japanese sika deer (Cervus nippon centralis) captured in Iwate Prefecture, Japan

Thirty-six flukes were collected from the livers of wild deer (Cervus nippon centralis) captured in Iwate Prefecture, Japan, and were served for morphometry. The length and/or the width of the body, suckers, testes, ovary, vitelline glands, cirrus pouch and eggs in the uterus of the flukes were measured. The distance between anterior end of the body and position of the maximal body-width or upper end of the testes were also determined. A remarked morphological characteristic was that the right and left testes did not lie tandem but lined bilaterally. Also the position of the maximal body-width did not always locate in the posterior part of the body of the fluke. The property was in accordance with those for Dicrocoelium chinensis.     

Morphology of the oviduct fluke,Prosthogonimus ovatus,isolated from Indonesian native chickens and histopathological observation of the infected chickens

Prosthogonimus ovatus infection was detected in 5 of 130 chickens in the oviduct and 4 chickens in the bursa of Fabricius. Scanning electron microscopy (SEM) revealed that the spines of the P. ovatus were densely distributed on the cuticula of the entire dorsal surface of body, but on the ventral surface, they were densely present to the level of ventral sucker but gradually decreased in density posteriorly, and they could not be seen in the posterior 1/3 area. The spines were finger-shaped and denticulate at the tip. Histopathological examination showed that polypous elevations, degeneration and exfoliation of the mucosal epithelium were detected in the bursa of Fabricius possibly by the suction of flukes, in addition to the stratification of the mucosal epithelium, and interstitial cell infiltration.     

Radiography and computed tomography of the heart and lower respiratory tract in toco toucans (Ramphastos toco)

This study aimed to evaluate normal features of the heart and lower respiratory tract in toco toucans by means of radiography and helical computed tomography (CT) scanner. Fifteen healthy adult toco toucans (Ramphastos toco), 10 females and 5 males, average body mass of 650 g were studied. CT examination as well as right lateral and ventrodorsal radiographic examinations of the coelomic cavity were performed under chemical restraint. Heart, lungs, air sacs, trachea and syrinx were analysed. The mean values of heart length, heart width and thoracic cavity in radiographs were, respectively, 23.76 mm, 25.94 mm and 48.87 mm. In both X-rays and CT scans, the lung parenchyma had honeycomb-like pattern. The topographic areas of the anterior and posterior air sacs were visualized as dark and air-filled spaces in X-rays. On CT evaluation, the air sacs occupied a larger area in the coelomic cavity compared to X-ray. In the lateral radiographic view, the cervical part of the trachea was positioned more ventrally in the transition from cervical to thoracic regions showing a V-shaped appearance. In all CT planes was visible division of the trachea into the right and left main bronchi at the level of 3rd thoracic vertebra. The syrinx was difficult to visualize in X-rays, but on CT it was easily identified in axial slice. In conclusion, the normal features of toco toucan's heart and lower respiratory tract that were determined on X-rays and CT scans are useful to compare with sick toco toucans, as well as other bird species.     

Fasciola hepatica: A comparative survey of adult fluke resistance to triclabendazole,nitroxynil and closantel on selected upland and lowland sheep farms in Northern Ireland using faecal egg counting,coproantigen ELISA testing and fluke histology

In order to investigate the incidence and distribution of adult fluke resistance to the fasciolicide tricalbendazole (TCBZ) amongst populations of Fasciola hepatica in sheep flocks in Northern Ireland (NI), individual rectal faeces samples were collected from 3 groups of 20 sheep, before (pre-dose), and 21 days after (post-dose) treatment of the animals with TCBZ, nitroxynil or closantel, on each of 13 well-managed sheep farms distributed across the province. The efficacy of each flukicide was determined for each farm, using faecal egg count reduction (FECRT) and F. hepatica coproantigen ELISA testing. In certain flocks, 2 sheep with high pre-dose faecal egg counts (FEC) were killed 3 days and 21 days respectively after TCBZ treatment, and the histology of the fluke reproductive organs was compared with that of flukes from untreated sheep, and from sheep treated with nitroxynil or closantel 2 days prior to death, using haematoxylin and eosin (H&E) staining and an in situ hybridisation method (TdT-mediated dUDP nick end labelling [TUNEL]) to demonstrate apoptosis. Results from FECRT revealed that in all flocks with a high fluke burden, TCBZ was ineffective in treating chronic fasciolosis, and this finding was generally supported by the results of the coproantigen reduction test (CRT). The histology of reproductive organs of flukes from TCBZ-treated sheep in these flocks was normal, when compared with untreated flukes, and this, together with the FECRT and CRT findings, indicated a likely diagnosis of TCBZ resistance in all the flocks with a high fluke burden. In contrast, nitroxynil and closantel were found to be fully effective against TCBZ-resistant flukes in each of the flocks bearing a high chronic fluke burden. All of the flocks with a high fluke burden and TCBZ resistance were managed on lowland in the South and East of NI. Upland flocks, in the North and West, had low fluke burdens, or were clear of infection; and FECs were too low to allow valid resistance testing. The study highlights the high level of penetration of TCBZ resistance throughout F. hepatica populations in areas of intensively managed sheep production with a high level of fluke challenge. Further, it emphasises the importance of pre-emptive chemotherapeutic action against chronic fasciolosis, using flukicides effective against the egg-producing adult flukes to minimise pasture contamination for the next season's lamb crop. This study also exemplifies the use of several complementary methods (FECRT; CRT; fluke histology; comparative anthelmintic efficacy testing) for confirmation of a diagnosis of fluke drug resistance.     

Digestive enzymes along digestive tract of a carnivorous fish Glyptosternum maculatum (Sisoridae,Siluriformes)

This study attempted to determine the presence, distribution and levels of digestive proteases, pepsin, amylase and lipase in the homogenates of digestive tract of a freshwater, cold‐adapted, carnivorous teleost of Tibet, Glyptosternum maculatum (n = 10, mean weight: 129.6 ± 28.9 g, mean length: 195.1 ± 16.6 mm, approximately 6 years old), at different temperatures and pH levels. The descending order of protease activity was as follows: stomach (pepsin, 16.16 ± 0.96 U/mg protein), anterior intestine (3.18 ± 0.25 U/mg protein), posterior intestine (1.76 ± 0.21 U/mg protein) and middle intestine (1.52 ± 0.23 U/mg protein). Amylase activity levels in descending order were as follows: anterior intestine (0.0062 ± 0.0007 U/mg protein), stomach (0.0032 ± 0.0009 U/mg protein), middle intestine (0.0023 ± 0.0005 U/mg protein) and posterior intestine (0.0023 ± 0.0004 U/mg protein). The highest lipase activity was found in the anterior intestine (0.83 ± 0.25 U/mg protein), followed by posterior intestine (0.51 ± 0.19 U/mg protein), middle intestine (0.48 ± 0.09 U/mg protein) and stomach (0.39 ± 0.10 U/mg protein). The optimal temperature and pH level for protease were 50 °C and 9.0–10.0, respectively, along the intestine. The pepsin optima in the stomach were 30 °C and 2.0 respectively. The optimal temperature for amylase was found to be 30 °C along the digestive tract and the optimal pH was 7.0 in the intestine and 6.0 in the stomach. The optimal temperature and pH levels of lipase were 30 °C, pH 6.0 for the stomach and 40–50 °C, pH 8.0 for the intestine, respectively. Hepato‐somatic index was 1.35 ± 0.2% and relative intestine length was 0.90 ± 0.19. The weight ratio of the stomach and intestine to body weight was 1.63 ± 0.43% and 1.57 ± 0.24% respectively.