Morphological and molecular description of Armillifer moniliformis larvae isolated from Sri Lankan brown palm civet (Paradoxurus montanus).

We report Armillifer moniliformis species infecting the endemic Sri Lankan brown palm civet (Paradoxurus montanus) from the Knuckles Range Forest Conservation Area, Sri Lanka. Larval stages of A. moniliformis were found during the postmortem of three civet cats found dead. Morphological studies were done by a light microscope and a scanning electron microscope (SEM). Histopathological examination was conducted using tissue samples obtained from the liver. For the molecular analysis, DNA was extracted from the isolated third-stage larvae. The NADH dehydrogenase subunit 5 (ND5) and the second internal transcribed spacer region (ITS-2), a portion of the large subunit nuclear ribosomal DNA (28S), a portion of 18S ribosomal rRNA gene (18S), and cytochrome c oxidase subunit 1 gene (COX1) were amplified using polymerase chain reaction (PCR). Excysted third-stage larvae were observed in the lungs, omentum, the pleural cavity, the abdominal cavity, and the surface of the spleen and the pericardium. Around 88 third-stage larvae were isolated from three civet cats. First-stage larvae in the liver were surrounded by outer fibrous layer over the inner germinal layer and filled with clear fluid. Slight hemorrhage, leukocyte infiltration, and mild hepatocellular degeneration in the liver were observed. The SEM examination indicated the unique oral apparatus comprises the oval-shaped mouth opening in between two pairs of curved, retractile hamuli. The sequences obtained for ND5, ITS-2, 28S, 18S, and COX1 were 301, 382, 325, 414, and 644 bp in length respectively. Morphology, sequence similarity search, sequence alignment, and phylogenetic analysis identified this parasite as A. moniliformis.

Molecular characterization of Fasciola jacksoni from wild elephants (Elephas maximus maximus) of Sri Lanka: a taxonomic evaluation.

Fasciola jacksoni is a significant contributor to the health and mortality of Asian elephants, particularly those in Sri Lanka. Despite the impact of fascioliasis on elephant populations, it is a neglected veterinary disease with limited taxonomic understanding. Molecular characterization and phylogenetic analysis of F. jacksoni were carried out to evaluate its suggested basal position in the Fasciolidae. Adult worms were collected during post-mortem of elephants, and eggs were collected from living elephants in National parks across Sri Lanka. Using the mitochondrial genes nicotinamide dehydrogenase subunit 1 (nad1) and cytochrome oxidase subunit 1 (cox1), and a partial 28S ribosomal DNA (28S rDNA), DNA sequences were generated from the F. jacksoni adult and egg material. Maximum likelihood (ML) phylogenetic analyses did not resolve F. jacksoni to be basal to the Fasciolidae. Furthermore, the ML analyses showed that the genus Fasciola was not monophyletic and that F. jacksoni was a sister species to the deer liver fluke Fascioloides magna. A clear framework is required to determine the taxonomic status of F. jacksoni and this current study provides the first detailed application of molecular techniques from multiple hosts across Sri Lanka with the production of reference DNA sequences for this important parasite.

Heart and liver are infected in fatal cases of dengue: three PCR based case studies.

BACKGROUND: Dengue is a global problem mainly in the tropics. Meticulous clinical management of cases has reduced the death rate significantly, but large numbers of people still succumb to severe complications of the infection. Presence of myocarditis is often overlooked leading to a poor outcome. Clinical management guidelines of dengue do not stress the importance of myocarditis as a manifestation in dengue infection. Severe hepatic dysfunction also needs emphasis. CASE PRESENTATION: We present three patients who had come to hospital on the 3rd day of fever. Two of them (case 1 and 3) were in shock on admission and case 2, who was stable on the3rd day, went into the critical phase and developed shock while in the hospital on the 4thday. All three had tachycardia on admission that got worse with time. The clinical course was unstable with fluctuations in urine output and deterioration of organ function. Despite frequent monitoring and life support they survived only 2-3 days in hospital. All three patients had myocarditis during the critical phase. In the first case, myocarditis was confirmed by troponin estimation and echocardiogram. In the second and third cases, histopathology confirmed myocarditis. Haemorrhagic necrosis of the liver was found in case 2 and 3 with exponential rise of transaminases. In all three cases, viral RNA was detected in both heart and liver tissues by PCR amplification. CONCLUSIONS: We stress that detection of myocarditis and liver involvement in any dengue patient is important from the onset of the illness where treatment should be tailored to prevent development of hypotension. Our findings are novel as PCR and histology are rarely done on tissues of deceased dengue patients in the world. Studies are needed to find therapeutic interventions to reverse cardiac and hepatic dysfunction in dengue infection.

Extensive haemorrhagic necrosis of liver is an unpredictable fatal complication in dengue infection: a postmortem study.

BACKGROUND: Dengue infection carries a potential risk of death despite stringent management of plasma leak and haemorrhage. It appears that the extent of liver dysfunction determines the outcome. METHODS: We present a postmortem study of five patients, died of dengue shock syndrome who had markedly elevated liver enzymes and irreparable circulatory failure. RESULTS: All were females with a median age of 46 years (range 20-50 years). All had positive NS1 and IgM. Clinically, one patient developed severe degree of hepatic encephalopathy whilst three patients developed uncontrollable bleeding manifestations. Dengue virus was detected in three liver specimens by reverse transcription PCR. Histology of the liver revealed massive necrosis with haemorrhages in these patients with evidence of micro and macrovesicular steatosis with significant periportal inflammatory infiltrate. No significant ischaemic changes or necrosis was observed in the other organs. CONCLUSIONS: Severe haemorrhagic necrosis of the liver was the cause of death in these patients probably due to direct viral infection. Predilection for severe liver disease remains unknown. Therefore, it is prudent to think beyond plasma leak as the main pathology of dengue infection and attempts should be made to develop other treatment modalities to prevent and manage unforeseen fatal complications of dengue infection.

Effect of Soil Temperature on Canine Soil-Transmitted Nematodes in Kandy District with the First Record of Hookworm, Ancylostoma tubaeforme from Sri Lanka.

PURPOSE: Soil-transmitted nematodes (STNs) are widespread in tropical and subtropical regions, particularly where the communities are socio-economically challenged. We investigated the effect of soil temperature on the prevalence and intensity of STN infection in free-roaming dogs. METHODS: Fresh faecal samples collected from free-roaming dogs in Digana and Pussellawa town areas in the Kandy District, Sri Lanka, were microscopically analysed for canine STNs. Soil temperature was measured at each sampling site. Highly prevalent canine hookworm Ancylostoma, was further studied using PCR and sequencing, followed by phylogenetic analysis. RESULTS: The soil temperature ranged between 28 and 31 °C (mean = 29.79 °C) and 18-21 °C (mean = 19.52 °C) in Digana and Pussellawa, respectively, showing a significant difference in the two sites (Students t-test t = 1.68, p < 0.0001). Of the total 44 dogs sampled, 41 (93.2%) were positive for STNs. During microscopic analysis, five nematodes: Ancylostoma spp., Capillaria sp., Strongyloides sp., Toxocara canis, and Trichuris sp., were identified. Ancylostoma species (93.2%) were the most prevalent, followed by Strongyloides sp. (22.7%) and Toxocara canis (15.9%). Infection prevalence of Strongyloides sp. was higher in Digana (40.9%) compared to that in Pussellawa (4.5%; Chi-square test, χ2 = 8.28, p = 0.004) and also the infection intensity from Digana (EPG = 8.02 ± 20.2) compared to that from Pussellawa (0.45 ± 2.1; Mann Whitney U test, p = 0.006). Amplicons (ITS1-5.8S-ITS2) of the expected size for A. caninum, and A. tubaeforme were produced. An A. caninum sequence reported here (OQ101719) illustrated the highest similarity of 99.2% to one of the local sequences (MZ707153) upon pairwise comparison. CONCLUSION: Digana, with a higher soil temperature than Pussellawa, had a significantly higher prevalence and infection intensity, particularly Strongyloides sp. This study also signifies the first molecular identification of hookworm species A. tubaeforme in Sri Lanka.

Morphology and surface topography of the schistosome Bivitellobilharzia nairi from the Asian elephant (Elephas maximus maximus) in Sri Lanka.

Bivitellobilharzia nairi was first recorded from an Indian elephant (Elephas maximus) in Berlin. Infections with this parasite have become increasingly important in E. maximus maximus populations in Sri Lanka. The present work is the first morphological description of this schistosome from Sri Lanka. A number of adult worms were recovered from a dead Asian elephant near the elephant orphanage, Pinnawala, in Sri Lanka. The observed clinical features of the infected elephant included emaciation, subventral oedema and anaemia. Post-mortem results indicated that the liver was enlarged and adult schistosomes were found in the blood vessels of the liver parenchyma. The total number of worms recovered from a portion of the liver was 129,870, which is an average of 22 worms per 100 g of liver. The present study uses both light microscopic and scanning electron microscope (SEM) techniques for the morphological and topographical characterization of this parasite and to permit comparison with other species of schistosomes. Morphologically, these worms correspond very well to the description of B. nairi by Dutt & Srivastava (1955). Moreover, it is clear that B. nairi is a distinctive species easily differentiated from other schistosomes. The SEM study of the tegument of male worms shows that the surface of B. nairi is smoother than in other schistosomes.

Polyphyly of the Dinurinae Looss, 1907 (Digenea: Hemiuridae) and resurrection of the Mecoderinae Skrjabin & Guschanskaja, 1954 based on novel collection of Tubulovesicula laticaudi Parukhin, 1969 from marine elapid snakes in Sri Lanka.

With one exception, the only known hemiurid trematodes that do not use teleost fishes as definitive hosts instead occur in marine elapid snakes. These comprise six species across four genera and three subfamilies, and so presumably indicate at least three independent invasions of marine snakes from teleost fishes. Here, one of these taxa, Tubulovesicula laticaudi Parukhin, 1969 (= T. orientalis Chattopadhyaya, 1970 n. syn.) is reported from Sri Lanka, collected from Shaw's sea snake Hydrophis curtus (Shaw) (Elapidae: Hydrophiinae: Hydrophinii), the annulated sea snake H. cyanocinctus Daudin and the yellow sea snake H. spiralis (Shaw) off Nayaru in the Bay of Bengal, and from H. spiralis in Portugal Bay, Gulf of Mannar. Novel molecular data, for COI mtDNA and ITS2 and 28S rDNA, are the first for a species of Tubulovesicula Yamaguti, 1934. Nominally, Tubulovesicula belongs in the Dinurinae Looss, 1907, but in phylogenetic analyses based on 28S rDNA, our sequences for T. laticaudi resolved relatively distant from that for representatives of Dinurus Looss, 1907, the type-genus, rendering the subfamily polyphyletic. Tubulovesicula laticaudi resolved closest to data for the type-species of the Plerurinae Gibson & Bray, 1979, but that subfamily is also polyphyletic. These findings lead us to re-evaluate an alternative classification considered by Gibson & Bray (1979). We propose restricting the Dinurinae for forms with a permanent sinus-organ (Dinurus, Ectenurus Looss, 1907; Erilepturus Woolcock, 1935; Paradinurus Vigueras, 1958; Qadriana Bilqees, 1971) and resurrect the Mecoderinae Skrjabin & Guschanskaja, 1954 for forms with a temporary sinus-organ (Mecoderus Manter, 1940, Allostomachicola Yamaguti, 1958, Stomachicola Yamaguti, 1934 and Tubulovesicula).

Immunolocalization of arginine kinase (AK) in Toxocara canis, Toxocara vitulorum, and Ascaris lumbricoides.

Arginine kinase (AK) is a member of the phosphagen kinase family. AK plays a major role in cellular energy metabolism in invertebrates including nematodes. In the present study, we performed the direct immunofluorescence test to determine the immunolocalization of AK in different stages of the life cycle (eggs, larvae, and adult worms) of Toxocara canis, Toxocara vitulorum, and Ascaris lumbricoides. Our results indicated variable levels of expression of AK in different stages. Moreover, strong fluorescence was observed in cleaving eggs than in dormant eggs. The highest activity of the enzyme was observed in the fully developed eggs. This may be due to high expression of AK in embryonic development, which is associated with increased energy demand due to cleavage and cellular differentiation. Surprisingly, expression of AK is significantly higher in the middle part and posterior end compared to anterior end of the larvae. In addition, AK is highly concentrated in cellular and metabolically active parts of the body such as hypodermis, muscle, intestine, ovaries, oviducts, and uterus, while it is absent in noncellular areas like cuticle. The present study revealed the presence of AK in T. canis, A. lumbricoides, and T. vitulorum and that it plays a major role in energy metabolism of these nematodes. Interestingly, antiserum was prepared against the recombinant T. canis AK and reacts with the native AKs of T. canis, A. lumbricoides, and T. vitulorum. AK levels could vary in relation to maximum potential rates of ATP turnover, oxidative capacity, and energy output. Further studies on subcellular localization of AK in these important helminths provide new information for researchers to develop effective anthelmintics against the parasites of veterinary and of public health importance.

First Molecular Identification of Ancylostoma Species in Dogs in a Rural Tea Estate Community in Sri Lanka and the Detection of Other Zoonotic Gastro-intestinal Parasites.

PURPOSE: Ancylostoma spp. causing canine hookworm infections are distributed worldwide with a high prevalence in the South Asian region. Here, we report the first molecular identification of the canine hookworm infecting the dogs in Sri Lanka. METHOD: Fresh faecal samples were collected from free-roaming dogs in tea estate communities in Talawakelle, processed and microscopically analysed for gastro-intestinal parasites. The common dog hookworm Ancylostoma sp. was further investigated using molecular diagnostic tools including PCR, sequencing, and phylogenetic analyses. RESULTS: Of the individual dogs sampled, 97.0% (97/100) were positive for GI parasites, identifying 14 species, all of which were zoonotic. Entamoeba sp. (58.0% (58/100)) was the most prevalent infection and Ancylostoma sp. (45.0% (45/100)) was the most prevalent helminth infection. Overall, helminth prevalence was higher in female dogs (92.5% (37/40)) than male dogs (73.3% (44/60)), potentially due to immune suppression during pregnancy and lactation. The prevalence and burden of Toxocara canis was higher in puppies (≤ 1 year) (68.8%(11/16); Chi-square test; χ2 = 20.912, p < 0.001 and FEC = 5.91; Mann-Whitney U test; p < 0.001), possibly due to vertical trans-mammary or trans-placental transmission of infective larvae. This study also presents the first record of Hymenolepis sp. from dogs in Sri Lanka. Ancylostoma sp. was molecularly confirmed as A. caninum. CONCLUSION: The local variant of A. caninum was phylogenetically unique and showed 99.2% similarity to that from India. The high prevalence of zoonotic GI parasites with public health significance highlights the importance of improving community well-being, veterinary care for dogs, and public awareness on zoonoses.

Comparative analysis of proximate compositions, mineral and functional chemical groups of 15 different seaweed species.

Seaweed is a popular edible source and is associated with many foods and pharmaceutical industries around the world. The current research aims to provide information on the chemical composition of 15 seaweed species, consisted of Chlorophyta, Ochrophyta/Phaeophyceae, and Rhodophyta macroalgae, collected from coastal areas of Sri Lanka. Seaweed samples were subjected to the analysis of lipids, proteins, ash and macro, micro, trace and ultra-trace elements. The highest protein content was recorded in the brown algae. Maximum dietary fiber and ash contents were recorded from green algae. The highest predominant fatty acids were observed from green seaweeds (Caulerpa racemosa); however, linoleic acid (C18:2n6) is the dominant fatty acid of all macroalgae. Mineral contents were highest in the red macroalga; however, copper, zinc and magnesium were also comparatively higher in green alga Ulva lactuca. In conclusion, 15 seaweed species belonging to the three different classes of seaweeds are investigated in details to obtain their biochemical, mineral and fatty acid compositions for the synthesis of novel therapeutic agents. In order to explore biorefinery processes for these seaweeds, as well as how they can potentially be cultivated, more extensive studies are required. Studying and determining the nutritional values of seaweeds will be beneficial with the potentials for future industrial uses and research.

The role of cortisol in the association of canine-companionship with blood pressure, glucose, and lipids: a systematic review.

INTRODUCTION: The dog is known as man's best friend and canine-companionship is associated with positive effects on cardiovascular health. AIM: We aim to review the role of cortisol in the association of canine-companionship with human blood pressure, glucose and lipid profile. METHODS: Electronic databases, and reference lists of the selected articles were searched for original articles in English which evaluate the role of cortisol in the association of canine-companionship with human blood pressure, glucose and lipid profile. Appropriate tools from the National Institute of Health were used for the quality assessment. RESULTS: Of the 2108 articles included for screening, 6 studies fulfilled the selection criteria. The USA had produced the highest number of studies (50%, 3/6). Pre-post studies (83%, 5/6) were the predominant type, and the overall quality of the selected studies was acceptable. The majority of studies showed a significant reduction of cortisol with dog companionship or therapy (67%, 4/6) along with a significant reduction of heart rate (2/4), systolic blood pressure (1/4), mean arterial blood pressure (1/4), or total cholesterol level (1/4). The role of cortisol in the association of canine-companionship with human blood pressure, glucose and lipid profile was scarcely studied with no studies from lower-middle-income countries, where the disease burden is on the rise. CONCLUSION: A significant reduction of few cardiovascular risk factors was found along with a significant reduction of cortisol in canine companionship in few studies. Future comparative or higher-level studies are essential on the association between canine companionship, cortisol and cardiovascular risk factors, especially in lower-middle-income countries.

Gastro-intestinal parasites in two subspecies of toque macaque (Macaca sinica) in Sri Lanka and their zoonotic potential.

Gastro-intestinal (GI) parasites of primates have a greater potential of becoming zoonotic. This potential may vary in different primates based on multiple factors such as proximity to human settlements and the climate of their habitat. We examined the GI parasites in two subspecies of toque macaque: Macaca sinica sinica (confined to the dry zone) and Macaca sinica aurifrons (confined to the wet zone) of Sri Lanka. Fresh faecal samples were collected and analysed following a modified Sheather's sucrose floatation method. A total of 90.8% (89/98) macaques were infected with one or more parasite species. There was no difference in the overall prevalence of GI parasites between the two subspecies, M. s. aurifrons (95.9%) and M. s. sinica (85.7%; χ2;χ = 3.059, p = 0.080). Sixteen parasite species were recorded including, 15 species in the M. s. sinica and 12 species in the M. s. aurifrons. Among the helminths identified, Anatrichosoma sp., Ancylostoma spp., Capillaria spp., Oesophagostomum /Bunostomum spp. and Physaloptera spp. are known to be zoonotic while Ascaris spp., Enterobius sp., Strongyloides spp. and Trichuris spp. have both zoonotic and anthroponotic potential. Among the protozoans, Balantidium coli and Buxtonella sp. are known to be zoonotic, while Entamoeba spp. and Cryptosporidium spp. have both anthroponotic and zoonotic potential. This study provides the first record of Anatrichosoma sp. and Buxtonella sp. in Sri Lanka and the first record of Cryptosporidium spp. in M. s. aurifrons. The molecular data allowed further identification and differentiation of Entamoeba nuttalli and E. coli that are known to be zoonotic and anthroponotic, respectively. The two subspecies of macaques have close interactions with humans; hence, in-depth epidemiological studies are required to understand the potential public-health risks to humans and conservation implications for macaque populations.

In vivo anticancer effects of Momordica charantia seed fat on hepatocellular carcinoma in a rat model.

BACKGROUND: Momordica charantia or bitter melon is a well-known vegetable with a number of therapeutic actions in Ayurvedic medicine. Alpha-eleostearic acid, a conjugated trienoic fatty acid present in bitter melon is proven to have anticancer properties. Crude seed oil from local bitter melon varieties could be an effective and economical anticancer therapy. OBJECTIVE(S): The study was conducted to evaluate the anticancer effect of the crude oil from the seeds of Matale green variety of bitter melon on a hepatocellular carcinoma-induced rat model. MATERIALS AND METHODS: Hepatocellular carcinoma (HCC) was experimentally induced in Wistar rats. Crude seed oil of Matale green bitter melon (MGBM) was supplemented to one treatment group in concurrence with carcinoma induction and to another treatment group after the development of carcinoma. After 168 days, gross morphological, histopathological, biochemical, hematological and gene-expression analysis of treated and control groups were performed. RESULTS: Oral supplementation of MGBM seed oil showed a statistically significant reduction (p < 0.05) in the average number, diameter and area of hepatic dysplastic nodules and a reduction in the size of histopathological neoplastic lesions in both treatment groups compared to the non-treated control group. The expression of tumor suppressor gene p53 and anti-apoptotic gene Bcl-2 were significantly increased while the expression of apoptotic gene caspase 3 was significantly reduced in the treatment group when MGBM supplementation was in concurrence with carcinogenesis (p < 0.05). CONCLUSION: Crude seed oil from the MGBM has anticancer effects against experimentally induced HCC in Wistar rats, specially when supplemented in concurrence with carcinoma induction.

Human Otoacariasis in Two Climatically Diverse Districts in Sri Lanka: Seasonality, Risk Factors, and Case Notes.

PURPOSE: Otoacariasis is a parasitic otopathy reported in many parts of the world. This study presents the seasonal pattern, risk factors, and case notes on human otoacariasis in two climatically different districts: Anuradhapura and Kandy in the Dry and Wet/Intermediate zone of Sri Lanka, respectively. METHODS: Ticks removed from the ear canal of patients were collected. Risk factors of otoacarisis were determined by a case/control follow-up study. RESULTS: Nymphal Dermacentor auratus (90.8%) was the main tick species associated otoacariasis. In the Kandy District, infestation was year-round, while in the Anuradhapura District, it was seasonal with a peak in December-February. Children < 10 years were a risk group in both districts. Females were a risk group in the Kandy District. Engagement in outdoor activities was a risk factor in both districts. In addition, the presence of wildlife and domesticated animals were risk factors in the Kandy and Anuradhapura districts, respectively. The treatment protocols in the two hospitals were different. An infant with otoacariasis from the Anuradhapura District developed rickettsia. CONCLUSIONS: Although the tick species was the same, seasonality, risk groups, and risk factors in the two districts were different which could be due to the establishment and persistence of tick populations influenced by biotic and abiotic factors.

In vitro and in vivo evaluation of the wound healing properties and safety assessment of two seaweeds (Sargassum ilicifolium and Ulva lactuca).

Seaweeds have been regarded as a reservoir of biologically active molecules that are important in the pharmaceutical industry. The aim of the present study was to explore the wound healing properties and to assess the safety of the seaweed Sargassum ilicifolium and Ulva lactuca. Enhanced cell proliferation and cell migration activities were observed in L929 cells treated with S. ilicifolium extract compared to U. lactuca extract treated cells and the control group. In-vivo experiments were conducted using five groups (10 in each) of Albino mice (BALB/c). Mice in group I and group II were treated (Orally, 100 mg/kg BW/day) with aqueous extracts of S. ilicifolium and U. lactuca, respectively for 14 days. Treatment group III received a topical application of the aqueous extract of S. ilicifolium (25% w/w) and ointment base (75% w/w) (2 g/kg BW/day, for 14 days). Group IV (Control) received an equal amount of distilled water, orally and mice in group V kept without wounds. The extract from S. ilicifolium showed stronger wound healing properties than the one from Ulva lactuca. Histopathological findings also revealed that the healing process was significantly enhanced in the mice group treated orally with S. ilicifolium aqueous extract. These findings show that S. ilicifolium species possess promising wound healing properties in-vitro and in-vivo.

Assessment of the Quality and Evaluation of the Antioxidant Potential of a Novel Sri Lankan Ayurvedic Polyherbal Formulation.

BACKGROUND: In Sri Lanka, a Polyherbal Ayurvedic Formulation (PHAF), which consists of powders of seven medicinal plants, is being trialed for use as an anti-inflammatory drug. In general, anti-inflammatory drugs have good antioxidant properties. Therefore, in the present study, an attempt was made to assess the quality and evaluate the antioxidant potential of PHAF. METHODS: Physicochemical parameters such as ash content, extractable matter, phytochemical screening for secondary metabolites, levels of heavy metals, and microbes were determined according to standard protocols. Antioxidant activity was evaluated using five in vitro assays: total polyphenolic content (TPC), total flavonoid content (TFC), ORAC (oxygen radical absorbance capacity), DPPH (1,1-diphenyl-2-picryl-hydrazyl), and ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt. RESULTS: PHAF contained 5.6 ± 0.2% of moisture, 6.5 ± 0.1% of total ash, 1.4 ± 0.1% of water soluble ash, 0.9 ± 0.0% of acid insoluble ash, 7.7 ± 0.2% of hot water extractable matter, 3.9 ± 0.1% of cold water extractable matter, 10.5 ± 4.1% of hot-ethanol extractable matter, and 8.4 ± 0.2% of cold-ethanol extractable matter. Phytochemical screening revealed the presence of phenolic compounds, tannins, flavonoids, coumarins, and saponins in both aqueous and ethanolic extracts of the drug. TPC, TFC, ORAC, DPPH, and ABTS of aqueous and ethanol extracts of PHAF were 103.65 ± 4.94 and 327.07 ± 9.65 mg gallic acid equivalents/g extract, 76.6 ± 5.83 and 224.6 ± 8.42 mg quercetin equivalents/g of extract, 481.11 ± 17.30 and 1481.44 ± 30.20 mg trolox equivalents/g of extract, 79.50 ± 4.42 and 227.17 ± 6.16 mg trolox equivalents/g of extract, and 198.20 ± 4.55 and 577.08 ± 5.48 mg trolox equivalents/g of extract, respectively. CONCLUSION: Ethanolic extract of PHAF is better than aqueous extract in terms of antioxidant properties.

Characterization and phylogenetic properties of the complete mitochondrial genome of Fascioloides jacksoni (syn. Fasciola jacksoni) support the suggested intergeneric change from Fasciola to Fascioloides (Platyhelminthes: Trematoda: Plagiorchiida).

Fascioloides jacksoni (syn. Fasciola jacksoni, Cobbold, 1869) (Platyhelminthes: Echinostomatoidea), is a liver fluke that causes severe morbidity and mortality of Asian elephants (Elephas maximus maximus). Understandings on molecular diagnosis, epidemiology, genetics and evolution of this flatworm are limited. In this study, we present the complete mitochondrial DNA (mt) sequence of 14,952 bp obtained from an individual fluke and comparative characterization of mitogenomic features with fasciolids, primarily, Fascioloides magna and other taxa in the superfamily Echinostomatoidea. Taxonomic relationship within and between Echinostomatoidea, Opisthorchioidea and Paramphistomoidea in the order Plagiorchiida, are also taxonomically considered. The complete circular mt molecule of Fas. jacksoni contained 12 protein-coding, two ribosomal RNA, 22 transfer RNA genes, and a non-coding region (NCR) rich in tandem repeat units. As common in digenean trematodes, Fas. jacksoni has the usual gene order, the absence of atp8 and the overlapped region by 40 bp between nad4L and nad4 genes. The NCR located between tRNAGlu (trnE) and cox3 contained nine nearly identical tandem repeat units (TRs of 113 bp each). Special DHU-arm missing tRNAs for Serine were found for both, tRNAS1(AGN) and tRNAS2(UCN). Base composition indicated that cox1 of Fas. jacksoni showed the lowest (11.8% to Fas. magna, 12.9 - 13.6% to Fasciola spp. and 18.1% to Fasciolopsis buski) and nad6 the highest divergence rate (19.2%, 23.8-26.5% and 27.2% to each fasciolid group), respectively. A clear bias in nucleotide composition, as of 61.68%, 62.88% and 61.54%, with a negative AT-skew of the corresponding values (-0.523, -0.225 and - 0.426) for PCGs, MRGs and mtDNA for Fas. jacksoni and likewise data for the fasciolids. Phylogenetic analysis confirmed the sister branch of Fas. jacksoni and Fas. magna with the nodal support of 100%, clearly separated from the taxonomically recognized Fasciola spp. With the previous studies, mitogenomic data presented in this study are strongly supportive for Fasciola jacksoni reappraisal as Fascioloides jacksoni in the Fascioloides genus.

Morphology and Molecular Characterization of Parabronema smithii (Cobbold, 1882) (Nematoda: Habronematidae) from Wild Asian Elephant (Elephas maximus maximus) of Sri Lanka.

PURPOSE: The aim of the present study was to carry out a detailed study of morphological features and to determine the phylogenetic position of Parabronema smithii (Cobbold, 1882) found in wild elephants in Sri Lanka. METHODS: Adult worms were collected from stomach ulcers at postmortem examination of wild elephants in the Udawalawe National Park, Sri Lanka. The detailed morphology of P. smithii was studied using light microscopy and, for the first time, scanning electron microscopy. Fifteen morphological characteristics were investigated. The phylogenetic analysis was conducted using the second internal transcribed spacer region (ITS2), and portions of the large subunit ribosomal DNA (28S) and cytochrome c oxidase subunit 1 (cox1). Furthermore, the present study provides a comparison of morphology and morphometrics of Parabronema species that occur in different hosts. CONCLUSION: Parabronema smithii isolated from wild elephants exhibited the key morphological features. Phylogenetic analysis of selected genes revealed that P. smithii is closely associated with P. skrjabini and Habronema spp. Findings of the present study enhance our understanding of the biology and taxonomy of P. smithii in wild elephant in Sri Lanka and will contribute to future phylogeographic studies.

Preliminary screening of the aqueous extracts of twenty-three different seaweed species in Sri Lanka with in-vitro and in-vivo assays.

BACKGROUND: Seaweeds are an important source of bioactive compounds which are applied in various aspects of medicinal investigations. The present study was conducted to investigate cytoxicity (in-vitro and in-vivo) and wound healing activity of different seaweed species in Sri Lanka. METHODS: Twenty-three seaweed samples, belonging to Phaeophyta (Brown), Chlorophyta (Green) and Rhodophyta (Red) were used for the experiments. Samples were collected from the inter-tidal and the sub-tidal habitats around Sri Lankan coast (Southern, Northern and North-western). Aqueous seaweed extracts were tested for cytotoxic and wound healing activity; in-vitro and in-vivo. To determine toxicity of aqueous seaweed extracts, brine shrimp lethality assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on mouse fibroblasts (L929) cell line were performed. Cell migration induction of seaweed extracts was assessed by scratch wound healing assay using L929 cell line. Based on the our previous experiments S.ilicifolium (SW23) was selected for the in vivo study to confirm our hypothesis. Albino mice (BALB/c) were divided into three groups (12 in each) and a circular area (44.07 ± 02.51 mm2) of full skin was excised to create a wound in mice group II and III. Group III received aqueous extract of Sargasum illicifolium (400 mg/kg BW/day for 12 days, orally), Group II received distilled water for 12 days whereas Group I was used as the control group and it was tested without forming wounds and without providing any treatment. Further, the expression level of Tumor Necrosis Factor (TNF-α) and Transforming Growth Factor-ß (TGF-ß) via RT-PCR were measured every three days until the end of the experiment. RESULTS: Phytochemical tests showed positive results to flavonoids in all the selected green seaweeds and alkaloids were observed in red seaweeds. In the toxicity assay, red seaweed, Acanthophora spicifera (SW17) was found to be highly effective on nauplii of brine shrimp (LC50 = 0.072 µg/µl). LC50 value of green seaweed species, Caulerpa racemosa (SW02 and SW08) and Caulerpa sertularioides (SW10) was not found within the tested concentration series. The highest cytotoxic effect on L929 cell line was exhibited by aqueous extracts of red seaweed; Jania adhaereus with 50.70 ± 7.304% cell viability compared with control group. The highest cell migration activity was observed in L929 cell line group treated with extracts of green seaweed namely; Halimeda opuntin (SW07) and extracts of brown seaweed namely; Stoechospermum polypodioides (SW11). Extracts of S. illicifolium (SW23) exhibited a significantly enhanced wound healing activity in mice group III within three days (P < 0.05) with an open wound area of 17.35 ± 1.94 mm2 compared with control group (26.29 ± 2.42 mm2). TGF-ß gene expression peaked on 6th day of post-wound and subsequently decreased on 9th day of post-wound in mice group III. TNF-α expression was suppressed in mice group III whereas it was elevated in group II. TGF-ß expression is enhanced in the treatment group compared to the control group. CONCLUSIONS: Aqueous extracts of selected seaweeds are a significant source of potential compounds with wound healing properties, which might be helpful in the healing of various wounds. This also infers that many species of brown and red seaweeds have the potential of wound healing, specifically, Sargasum illicifolium and Jania adhaereus could be a potential candidate for in-vivo studies related to wound healing and cancer therapy in the near future.

Toxocara canis and Toxocara vitulorum: molecular characterization, discrimination, and phylogenetic analysis based on mitochondrial (ATP synthase subunit 6 and 12S) and nuclear ribosomal (ITS-2 and 28S) genes.

Toxocara canis and Toxocara vitulorum are two important parasites of dogs and buffaloes with public health concern. The objectives of the present study are to identify molecular markers to discriminate these closely related parasites and to determine their phylogenetic position and genetic diversity within the genus Toxocara. Thus, two mitochondrial genes (complete ATPase 6 and partial small subunit ribosomal RNA (12S rDNA)), two nuclear ribosomal genes (second internal transcribed spacer region (ITS-2)), and part of the large subunit 28S region were analyzed. Nucleotide sequence (597 bp) and predicted amino acid sequences of the complete ATPase 6 gene (199 amino acids) of both species (T. canis and T. vitulorum) are similar in size with the Toxocara cati and Toxocara malaysiensis. There was 88% nucleotide similarity between T. canis and T. vitulorum and many transversions present in the 12S gene. Analyses of the ITS-2 and 28S regions revealed that the 28S region was more conserved (95% nucleotide similarity between T. canis and T. vitulorum) than the ITS-2 region (85%). This study has provided useful molecular markers for the molecular epidemiological investigation of Toxocara species. Further, phylogenetic analyses of the ITS-2 and 28S genes have indicated that the members of the genus Toxocara form a distinct group with reference to their definitive hosts.