In vitro screening as an anthelmintic discovery pipeline for Calicophoron daubneyi: nutritive media and rumen environment-based approaches.

Paramphistomosis can lead to morbidity and mortality of ruminant livestock within tropical and sub-tropical climates. In recent decades, rumen fluke has become an emerging infection in temperate climates across Western Europe, with Calicophoron daubneyi, the primary species present. Clinical outbreaks with C. daubneyi larvae are reported and adults might be responsible for production losses. There is not currently a widely licensed anthelmintic product available to control C. daubneyi. In this study, three existing flukicide anthelmintics were tested for efficacy against mature C. daubneyi, comparing a standard in vitro culturing assay and a new more relevant rumen fluid based in vitro compound screening protocol. The new rumen based screen confirmed that oxyclozanide was active against adult C. daubneyi and identified activity with praziquantel. The study highlighted the downstream value of incorporating relevant in vitro screening for anthelmintic discovery pipelines.

Cotylophoron marajoensis n. sp. (Digenea: Paramphistomidae) a parasite of Bubalus bubalis on Marajó Island, Pará, Brazilian Amazon.

The genus Cotylophoron belongs to the Paramphistomidae family and its definitive hosts are ruminants in general. This work describes the presence of a new species of the gender, a parasite in the rumen and reticulum of Bubalus bubalis, on Marajó Island in the Eastern Brazilian Amazon, using of light microscopy, scanning electronic microscopy and molecular biology techniques. One hundred and ten animals were analyzed, of which 4.54% were parasitized by flukes in their adult forms. The helminths were found fixed to the ruminal mucosa and present Liorchis-type pharynx, Cotylophoron-type genital sucker, oblique testicles larger than the ovary, uterus in rings full of eggs and Cotylophoron-type acetabulum. These morphologic characters do not fit into any previously described species. Thus, it is proposed that this is a new species in the genus Cotylophoron. The present work expands the record of parasitism by helminths in Bubalus bubalis, this being the first record of trematoda from the genus Cotylophoron for this host in the Brazilian Amazon.

Cotylophoron marajoensis n. sp. (Digenea: Paramphistomidae) a parasite of Bubalus bubalis on Marajó Island, Pará, Brazilian Amazon / Cotylophoron marajoensis n. sp. (Digenea: Paramphistomidae) parasito de Bubalus bubalis na Ilha de Marajó, Pará, Amazônia Brasileira

Abstract The genus Cotylophoron belongs to the Paramphistomidae family and its definitive hosts are ruminants in general. This work describes the presence of a new species of the gender, a parasite in the rumen and reticulum of Bubalus bubalis, on Marajó Island in the Eastern Brazilian Amazon, using of light microscopy, scanning electronic microscopy and molecular biology techniques. One hundred and ten animals were analyzed, of which 4.54% were parasitized by flukes in their adult forms. The helminths were found fixed to the ruminal mucosa and present Liorchis-type pharynx, Cotylophoron-type genital sucker, oblique testicles larger than the ovary, uterus in rings full of eggs and Cotylophoron-type acetabulum. These morphologic characters do not fit into any previously described species. Thus, it is proposed that this is a new species in the genus Cotylophoron. The present work expands the record of parasitism by helminths in Bubalus bubalis, this being the first record of trematoda from the genus Cotylophoron for this host in the Brazilian Amazon.

Resumo O gênero Cotylophoron pertence à família Paramphistomidae e possui como hospedeiros definitivos ruminantes em geral. Este trabalho descreve a presença de uma espécie nova do gênero, parasito do rúmen e retículo de Bubalus bubalis, na Ilha de Marajó, Amazônia oriental brasileira, a partir das técnicas de microscopia de luz, microscopia eletrônica de varredura e biologia molecular. Foram analisados 110 animais, dos quais 4,54% estavam parasitados por trematódeos na sua forma adulta. Os helmintos foram encontrados fixados à mucosa ruminal, apresentando faringe do tipo Liorchis, ventosa genital do tipo Cotylophoron, testículos oblíquos maiores que o ovário, útero em alças repleto de ovos, e acetábulo do tipo Cotylophoron. Estes caracteres morfológicos não se enquadram em nenhuma espécie previamente descrita. Assim, propõe-se uma nova espécie ao gênero Cotylophoron. O presente trabalho amplia o registro do parasitismo por helmintos em Bubalus bubalis, sendo este o primeiro registro de trematódeos do gênero Cotylophoron nesse hospedeiro para a Amazônia brasileira.

An in vitro confirmation of the ethonopharmacological use of Senna plants as anthelmintic against rumen fluke Paramphistomum gracile.

BACKGROUND: Paramphistomosis is a pathogenic disease of domesticated ruminants, causing great economic loss in dairy industry and meat production. It is considered as a neglected tropical disease with highest prevalence throughout tropical and subtropical regions, particularly in Africa, Asia, Europe, and Australia. There are few trematocidal drugs available in the market. Most are resistant and have elevated side effects. Therefore, alternative trematocidal drugs need to discover. This study was conducted to evaluate three plants leaf extracts (from Senna alata, S. alexandrina, and S. occidentalis) as effective remedies against gastrointestinal trematode parasite (Paramphistomum gracile) of ruminants. Live adult parasites were collected in 0.1 M phosphate-buffered saline (PBS) from fresh autopsied goat's rumen. Parasites were incubated in leaf extracts of S. alata, S. alexandrina and, S. occidentalis individually and in combination (1:1) ratio at 37 ± 1°C. Treatment media contain extracts at different concentrations (10, 20 and 40 mg/mL) in 10 mL of 0.1 M PBS with 1% dimethylsulphoxide (DMSO). Parasites in control group were incubated in PBS without extract. The efficacy of three Senna extracts was evaluated on the basis of dose-dependent motility and mortality of the trematode. Immediately after paralysis, all treated parasites were collected for histology, SEM and biochemical study. RESULTS: Dose-dependent efficacy was observed in terms of motility and time of mortality in all treated parasites after exposure in various concentrations of the Senna plant extracts. S. occidentalis and S. alexandrina showed better efficacy in combination than comparing with individual treatment groups. Histological study and scanning electron microscopic observations revealed conspicuous deformity of surface architecture in all treated parasites. Scanning electron micrographs also revealed shrinkage, vacuolization, infoldings and blebbings on the body surface of treated worms. Activities of tegumental enzymes were inhibited in all treatment groups compared to control. CONCLUSION: The overall findings from this study revealed that all three Senna leaf extracts individually and in combination showed potential antitrematocidal activity against Paramphistomum gracile by damaging body tegument and neural propagation. Thus, this study confirmed that all three Senna extracts can be considered as a potential drug-like candidate in indigenous system of traditional medicine against trematode infections in livestock.

In vitro anthelmintic effect of biologically synthesized silver nanoparticles on liver amphistome, Gigantocotyle explanatum.

In order to ensure global food security a rationale approach is required to control all those factors which directly or indirectly affect the food productivity. The neglected helminthic diseases alone are responsible for huge economic losses to the agrarian stakeholders. The problem is further compounded by the emerging drug resistance in flukes against the commonly used anthelmintics like triclabendazole. Therefore, the search for alternatives including the nano-based approaches has become a necessity to develop future control strategies. In the present study the effect of biologically synthesized silver nanoparticles (AgNPs) was investigated on an economically important amphistome parasite, Gigantocotyle explanatum, obtained from the infected liver of the Indian water buffaloes, Bubalus bubalis. In vitro treatment of the adult worms with different doses of AgNPs severely affected the worm motility and caused ROS mediated damages in the treated flukes. The antioxidant system and the detoxification ability of the worms appeared to be disrupted along with pronounced DNA damage in the treated worms as compared to the controls. Following the treatment of worms with different concentrations of AgNPs there was a significant (p < 0.05) increase in lipid peroxidation and protein carbonylation levels which are the key oxidative stress markers. The tegumental surface which is metabolically active, was severely damaged as evident from the loss of papillae, severe blebbing, shearing and erosion of the surface structures. Such topographical disruptions would facilitate the penetration of the nanoparticles deep within the tissues that might greatly reduce the invasive potential of the flukes as evident from the decreased motility. Taken together our findings suggest that the AgNPs posses great anthelmintic potential and could be further exploited for the development of anthelmintic formulations which may be tested in vivo.

Paramphistomum cervi: the in vitro effect of plumbagin on motility, survival and tegument structure.

Paramphistomiasis causes enteritis and anemia in livestocks and result in substantial production and economic losses. It is considered a neglected tropical disease, with no effective trematodicidal compound for treatment. Plumbagin (PB), a compound founds to be rich in the roots of Plumbago indica, is a naphthoquinone derivatives which can induce oxidative stress in parasites. In this study we have evaluated the anthelmintic activity of PB against adult Paramphistomum cervi by incubating the parasites in M-199 medium containing 0.1, 1.0, 10 and 100 µg/ml of the PB, and albendazole (ABZ) at the concentration of 100 µg/ml as the positive control, for 3, 6, 12 and 24 h, using relative motility (RM) assay and observed by scanning electron microscopy (SEM). After 12 h exposure with 100 µg/ml ABZ, flukes showed decreased contraction and motility. At 24 h incubation they showed only active movement of some part of the body. The PB-treated flukes at all concentrations showed rapid decrease of motility at 3 h incubation. In 0.1, 1.0 and 10 µg/ml of PB, the RM values were decreased sharply from 3 to 12 h, and then they were killed since 12 h in the incubation with 10 µg/ml of PB. The highest parasite mortality was found as early as 3h when they were incubated with 100 µg/ml of PB. The morphological changes on the tegumental surface were similar in both flukes treated with ABZ and PB, which sequentially comprised of swelling, followed by blebbings that later ruptured, leading to the erosion and desquamation of the tegument syncytium. As the result, lesions were formed which exposed the basal lamina. The damage appeared more severe on the ventral than the dorsal surface, and earlier on the anterior part and lateral margins of middle third when compared to the posterior part of the parasites's bodies. The severity and rapidity of the damages were enhanced with increasing concentration of PB, which showed stronger activity than ABZ. Hence, PB has a potential to be an anthelmintic drug against adult P.cervi.

Analysis of the tegument of Zygocotyle lunata (Trematoda: Paramphistomidae) adults by scanning electron microscopy.

Paramphistomiasis is a parasitic disease caused by various species of the Paramphistomidae. These species mainly affect domestic and wild ruminants; the economic impact of these diseases is often underestimated. Traditionally, the identification of paramphistomes has been difficult and has been based on morphological aspects such as the body shape, the position of the esophagus and cecae, or the tegumental papillae. Despite the many investigations regarding the tegumental papillae for other paramphistomes, very few efforts have been made using Zygocotyle lunata, partly because many authors differ with respect to the importance of papillae in the classification of paramphistomids. Herein, we characterize by scanning electron microscopy new tegumental papillae not previously described on the tegument of 3-wk-old Z. lunata adults obtained from mice. Three morphologically different papillae (rosette, ciliated, and conical papillae) were observed and are described. Based on these results, we assert that the newly recognized morphological features should be used for species differentiation in the future in addition to the new molecular techniques.

Fischoederius cobboldi: A scanning electron microscopy investigation of surface morphology of adult rumen fluke.

Adults Fischoederius cobboldi are conical-shaped, concave ventrally and convex dorsally, measures about 8-10mm in length and 4-6mm in width across the mid section. Scanning electron microscopy (SEM) of entire body showed that the tegumental surface exhibits highly corrugation and transverse folds alternating with grooves and without spines. At higher magnification, the surface of each fold is further increased with a meshwork of ridges separated by irregular-sized pits. The ventral surface has more complex corrugations and invaginations than those of the dorsal surface of the body. Both anterior and posterior suckers have thick edges covered with transverse folds and appear spineless. The genital pore is located at the anterior one-third of the body. There are two types of sensory papillae on the surface: type 1 is bulbous in shape and nipple-like tips, measuring 10-15 µm in diameter at the base, and also type 2 is a similar shape and has short cilia on tips. These sensory papillae occur in large clusters, each having between 7 and 25 units depending on the region of the body. Clusters of papillae on the ventral surface and around the anterior suckers tend to be more abundant and larger in size. The dorsal side of the body exhibit similar surface features, but papillae appear less numerous and are smaller. Corrugations and invaginations of the dorsal aspect are also less extensive than those on the ventral surface of the body.

Anthelmintic effect of a methanol extract of leaves of Dregea volubilis on Paramphistomum explanatum.

Dregea volubilis (family Asclepediaceae) is widely used as anthelmintic in traditional system of medicine in eastern and southern part of India. The aim of this study was to evaluate the anthelmintic activity of the methanol extract of D. volubilis leaves (MEDV) and to observe its effect through SEM study. Live parasites (trematode Paramphistomum explanatum) were collected from buffalo in 0.9% phosphate-buffered saline (PBS). It was incubated at 37 ± 1°C in media containing either no extract (control), the test drug, MEDV at four dose levels (10, 25, 50, and 100 mg/ml) or the standard drug, albendazole, at a dose of 10 mg/ml. The effectiveness of the extract was judged on the basis of the loss of spontaneous movement and/or complete destruction or death of the trematodes. After being removed from the experimental medium, trematodes were dipped in PBS at 37 ± 1°C and on gentle stimulation, the paralyzed parasite showed immobility. Death was confirmed when it completely lost its motility even when vigorously shaken or dipped in warm water (50°C). The trematodes, both drug treated and others, were further processed for SEM study using standard method. The anthelmintic activity was found with all the doses through paralysis and death of the organisms (p < 0.001). Maximum anthelmintic activity was found with a dose of 100 mg/ml. Minor damage was observed with both suckers, but severe distortion was found with tegumental surface of the treated trematodes. The present SEM-based study established the anthelmintic activity of MEDV.

Anthelmintic effect of a methanol extract of Bombax malabaricum leaves on Paramphistomum explanatum.

Bombax malabaricum (family Bombacaceae) is used as anthelmintic in traditional system of medicine in Southern Punjab of Pakistan. The objective of this study was to evaluate the anthelmintic activity of the methanol extract of B. malabaricum leaves (MEBM). Live parasites (trematode: Paramphistomum explanatum) were collected from buffalo in 0.9% phosphate-buffered saline. It was incubated in Petri dishes at 37 ± 1°C in media containing either no extract (control) or MEBM, the test drug at 10, 25, 50, and 100 mg/ml dose level or albendazole, the standard drug at 10 mg/ml. The efficacy of the extract or albendazole was measured on the basis of the loss of spontaneous movement and/or death of the trematodes. Paralysis was considered when there is no movement unless shaken vigorously. Death was confirmed when the trematodes completely lost their motility, even when vigorously shaken or dipped in warm water (50°C), followed by fading away of their body color. The trematodes, both drug treated and others, were further processed for SEM study using the standard method. All trematodes died with all the above-mentioned doses of MEBM within a short period of time (less than 45 min) which was statistically highly significant (p < 0.001). MEBM at 100 mg/ml showed maximum efficacy. It paralyzed and killed trematodes in 18.50 ± 0.62 and 22.17 ± 0.48 min, respectively. SEM study showed that MEBM-treated trematodes were stretched. The study established the anthelmintic activity of MEBM.

Paramphistomum cervi: surface topography of the tegument of adult fluke.

Adult Paramphistomum cervi or rumen fluke are pear-shaped, slightly concave ventrally and convex dorsally. The worm measures about 5-13 mm in length and 2-5 mm in width across the mid-section. As observed by scanning electron microscopy (SEM), the tegumental surface in all part of the body, appears highly corrugated with transverse folds alternating with grooves and is spineless. At high magnification, the surface of the fold is composed of microfolds or ridges separated by microgrooves or pits. Corrugations and invaginations of the ventral surface are also more extensive than on the dorsal surface of the body. Both anterior and posterior suckers have thick rims covered with transverse folds without spine. The genital pore is situated at the anterior third of the body. There are two types of sensory papillae on the surface: type 1 is bulbous in shape, measuring 10-15 microm in diameter at the base with nipple-like tips, and type 2 has a similar shape and size and also a short cilia on top. These sensory papillae usually occur in large clusters, each having between 5 and 20 units depending on the region of the body. Clusters of papillae on the ventral surface and around the anterior suckers tend to be more numerous and larger in size. The dorsal surface of the body has the least number of papillae.

Tegumental surface changes in adult Paramphistomum microbothrium (Fischoeder 1901) following in vitro administration of artemether.

The treatment of paramphistomiasis, a neglected tropical disease, has been carried out with different fasciolicidal compounds, all showing weak efficacy. Therefore, the search for alternative paramphistomicidal drugs is warranted. In the present study, the in vitro effects of artemether on adult Paramphistomum microbothrium were evaluated, for the first time, using scanning electron microscopy. After 24 h of incubation with 10 microg ml(-1) artemether, tegumental damage of both anterior and posterior ends of the fluke had occurred in the majority of the specimens examined. Sensory papillae surrounding the oral aperture were ruptured, while those at the acetabular region appeared to be sunken due to tegumental swelling. The tegumental disruption became more pronounced and both oral sucker and acetabulum were severely distorted, on increasing the concentration to 20 microg ml(-1). With higher concentration of 30 microg ml(-1), gross swellings of the body of the fluke, clearly visible to the naked eye, were observed, and damage to both oral sucker and acetabulum was so extreme that little recognizable structure remained.

Spermiogenesis in two paramphistomes from Nile fish in Egypt: an ultrastructural study.

The process of spermiogenesis in two paramphistomes, Sandonia sudanensis and Basidiodiscus ectorchis from the Nile fish Synodontis schall in Egypt was studied by transmission electron microscopy. Spermiogenesis is characterized by the outgrowth of the zone of differentiation, presenting two basal bodies separated by a microtubule organizing centre, each basal body developing into a flagellum. Proximodistal fusion of these flagella with a median cytoplasmic extension gives rise to the spermatozoon. The mature spermatozoon possesses two axonemes of the 9+'1' pattern typical of parasitic helminths. There are few ultrastructural studies on spermiogenesis in paramphistomes, which are considered the most primitive digenetic trematodes. The present study provides new and more detailed information on this process, including the presence of a lateral flange and external ornamentation of the cell membrane. The value of sperm ultrastructure as a taxonomic tool in phylogeny is also discussed.

Ultrastructure of spermiogenesis and the spermatozoon of Paramphistomum microbothrium [Fischoeder 1901] (Digenea: Paramphistomidae), a parasite of Bos taurus in Senegal.

The ultrastructural study of spermiogenesis and the spermatozoon of Paramphistomum microbothrium gave evidence of some characteristics of this digenean. The intercentriolar body located between two striated roots and two centrioles exhibits a symmetric plane. The two external bands of this intercentriolar body are made up of a line of granules. The striated roots are about 3.7 microm long. During spermiogenesis, a flagellar rotation of 90 degrees is described. The spermatozoon is characterized in its anterior part by the existence of external ornamentations and a lateral expansion exhibiting a "spine-like body". Such a spine-like body has never been described before in a digenean. The region II of the spermatozoon is asymmetrical and also characterized by the absence of mitochondrion. The posterior end of this spermatozoon exhibits a nucleus and a few microtubules.

Ultrastructure of spermiogenesis and the spermatozoon of Paramphistomum microbothrium (Fischoeder 1901; Digenea, Paramphistomidae), a parasite of Bos taurus in Senegal.

The ultrastructural study of spermiogenesis and the spermatozoon of Paramphistomum microbothrium gave evidence of some characteristics of this digenean. The intercentriolar body located between two striated roots and two centrioles exhibits a symmetric plane. The two external bands of this intercentriolar body are made up of a line of granules. The striated roots are about 3.7-microm long. During spermiogenesis, a flagellar rotation of 90 degrees is described. The spermatozoon is characterized in its anterior part by the existence of external ornamentations and a lateral expansion exhibiting a "spine-like body." Such a spine-like body has never been described before in a digenean. The region II of the spermatozoon is asymmetrical and also characterized by the absence of mitochondrion. The posterior end of this spermatozoon exhibits a nucleus and a few microtubules.

Breve descripción de Taxorchis schistocotyle Beddard, 1914 (Trematoda, cladorchiidae) por microscopia electrónica de barrido / Short description of Taxorchis schistocotyle Beddard, 1914 (Trematoda, cladorchiidae) with scanning electron microscope

The study describes some morphological trait of Taxorchis schistocotyle Beddard, 1914 (Trematoda, Cladorcniidae) using scanning electron microscope (SEM). Specimens were removed from small instestine during necropsy of Hydrochoeris hydrochaeris (Rodentia, Hydrocheoridae). Samples were fixed in glutaraldehid 3 percent in sodium monoacid phosphate buffer with dehidratation in acetones of increasing graduation. Diagnosis technique used was useful to survey and differentiation among species that parasite Argentinean wild mammalians.

In vitro anthelmintic activity of root-tuber extract of Flemingia vestita, an indigenous plant in Shillong, India.

The in vitro activity of root-tuber-peel extract of Flemingia vestita, an indigenous plant consumed by the natives in Northeast India, was tested against helminth parasites. Live parasites (nematode: Ascaris suum from pigs, A. lumbricoides from humans, Ascaridia galli and Heterakis gallinarum from domestic fowl; cestode: Raillietina echinobothrida from domestic fowl; trematode: Paramphistomum sp. from cattle) were collected in 0.9 % physiological buffered saline (PBS) and maintained at 37 +/- 1 degrees C. In vitro treatment of the parasites with the crude extract (50 mg/ml) in PBS revealed complete immobilization of the trematode and cestode in about 43 and 20 min, respectively. However, the cuticle-covered nematodes did not show any change in physical activity and remained viable even after a long period of exposure to the extract. Exposure of R. echinobothrida to genistein (0.5 mg/ml), an active principle isolated from the root-tuber peel, caused spontaneous loss of movement (paralysis) in 4.5 h, which was slower than the time required for praziquantel, the reference flukicide and cestodicide. The treated parasites showed structural alteration in their tegumental architecture. This study suggests the vermifugal activity of this plant extract against trematodes and cestodes.

Ultrastructure and histochemistry of the tegument of juvenile paramphistomes during migration in Indian ruminants.

The tegument of juvenile Paramphistomum epiclitum and Fischoederius elongatus (Paramphistomidae: Digenea) resembles those of other digeneans. Seven types of papillae were observed, mostly on the oral and acetabular surfaces, and increase in number during migration. Also evident are two types of secretory body (T1 and T2) which are synthesized separately in tegumental cytons underlying the syncytium. Exocytosis of T2 bodies occurs at the apical membrane and appears to contribute to a fibrous glycocalyx. The tegumental syncytium lining the pharynx and acetabulum is thinner and has a higher capacity for vacuolation than the general tegument. These may represent important sites for osmoregulation. The absence of mitochondria from the tegument in migrating juveniles suggests limited involvement in energy demanding processes. Pigmentation of the subtegument is first evident in mature cercariae and is progressively eliminated during migration.

Ultrastructure and histochemistry of the digestive tract of juvenile Paramphistomum epiclitum (Paramphistomidae: Digenea) during migration in Indian ruminants.

The digestive tract of juvenile Paramphistomum epiclitum consists of a foregut with a highly muscular terminal pharynx and an oesophagus, which leads to a pair of unbranched and blind-ending intestinal caeca. A syncytium lining the foregut is continuous with the external tegument and displays similar sensory papillae and secretory bodies (T1 and T2). A third type of secretory body (T3) is confined to the oesophageal cytons of newly excysted juveniles and is first evident in the syncytium by day 14 of migration. An epithelium lining the caeca is composed of a single layer of morphologically uniform cells whose apical surface is amplified by microvilli. Dense secretions synthesized in the caecal epithelium of mature cercariae are released during migration by a mechanism resembling modified apocrine discharge. The caecal epithelium of migrating juveniles undergoes a 10-fold increase in surface amplification (irrespective of growth) during its transition from a primarily secretory tissue to one apparently specialized for absorption.