Time-dependent tegumental surface changes in juvenile Fasciola gigantica in response to triclabendazole treatment in goat.

Triclabendazole (TCBZ), the anthelmintic drug active against both mature and immature liver flukes, was used to investigate the effect of in vivo treatment on the tegumental surface of juvenile Fasciola gigantica. Five goats were infected with 150 F. gigantica metacercariae each by oral gavage. Four of them were treated with single dose of TCBZ at 10mg/kg at four weeks post-infection. They were euthanized at 0 (untreated), 24, 48, 72 and 96h post treatment. Juvenile flukes were manually retrieved from the goat livers and processed for scanning electron microscopy. In control flukes, the anterior region was adorned with sharply pointed spines projecting away from the surface, while in the posterior region, spines become shorter and narrower, loosing serration and with the appearance of distinct furrows and papillae. The dorsal surface retained the same pattern of surface architecture similar to that of ventral surface. Flukes obtained from 24h post-treatment did not show any apparent change and were still very active. However, there were limited movements and some blebbing, swelling, deposition of tegumental secretions and some flattening displayed by the flukes of 48h post-treatment. All the worms were found dead 72h post-treatment and showed advanced level of tegumental disruptions, consisting of severe distortion of spines, sloughing off the tegument to expose the basal lamina, formation of pores and isolated patches of lesions. By 96h post-treatment, the disruption was extremely severe and the tegument was completely sheared off causing deeper lesions that exposed the underlying musculature. The disruption was more severe at posterior than anterior region and on ventral than dorsal surface. The present study further establishes the time-course of TCBZ action in vivo with 100% efficacy against the juvenile tropical liver fluke.

A rabbit eye model for in vivo transformation of progenetic metacercariae of Clinostomum complanatum into ovigerous adult worms.

Clinostomum complanatum is a digenetic trematode that causes yellow grub disease in some fish species and also shows zoonotic potential by sporadically infecting humans. In this study, progenetic metacercariae of C. complanatum were obtained from the fish Trichogaster fasciatus, and were aseptically placed in conjunctival incisions made in the superior and inferior fornices of the eye of rabbits, which served as the experimental hosts. Worms were harvested without necropsy of the host on days 4 and 8 post infection, to observe in vivo transformation of the progenetic metacercariae into ovigerous adult worms. The worms appeared to cause minimal damage to the host although they were tenaciously attached. In vivo maturation was evident by the development of the vitellaria, enlargement of gonads, the presence of a large number of shelled eggs in a distended uterus and ramifications of the intestinal caeca. Obtaining mature ovigerous worms without sacrificing the host clearly gives the rabbit eye model an advantage over those described previously. Due to the relative advantage of the short time required for maturation and the prolific egg production by C. complanatum, it is suggested that this host-parasite system could be used as an excellent model for classroom teaching of trematode biology and to investigate the cues involved in in vivo transformation and host-parasite interactions.

Diagnostic evaluation of supraclavicular lymphadenopathy.

Patients presented with the supraclavicular lymphadenopathy in the medicine department have a strong suspicion of serious illness like tuberculosis, sarcoidosis, toxoplasmosis and malignancy of lymphnode, blood, lung, upper GIT, breast, ovary, testes, and other sites of body. This prospective type of observational study carried out in the indoor and out patient department of medicine of Mymensingh Medical College Hospital over a period of 6 month from April 2011 to September 2011 to diagnose the causes of supraclavicular lymphadenopathy. Patient of either sex, 18 years or above presented with supraclavicular lymphadenopathy were included. Biopsy or FNAC were done. The study showed that mean age of the patient of supraclavicular lymphadenopathy that finally diagnosed as malignant was 49.7 years and that of non malignant was 33.7 years. Male patient have suffered more (60%) from malignant disease than that of female patient (40%). Discrete, hard, non tender either fixed or non fixed supraclavicular lymphadenopathy was found malignant (18 of 18 cases, 100%) and discrete, firm, tender lymphnode were found non malignant (5 of 5 cases, 100%). Increased frequency (11 of 28, 39.3%) of granulomatous inflammation from the tuberculoid lymphadenitis were found among the patient undergone supraclavicular lymphnode biopsy. FNAC result was also of simillar type and finally it was found that frequency of tuberculosis (20 of 53, 37.7%) was highest and bronchial carcima was the second most frequent diagnosis (14 of 53, 26.4%). This study showed that supraclavicular lymphadenopathy is associated mostly with serious disease like tuberculosis and malignancy.

Characterization of the glutamate dehydrogenase activity of Gigantocotyle explanatum and Gastrothylax crumenifer (Trematoda: Digenea).

Glutamate dehydrogenase (GLDH) (EC 1.4.1.3) is a ubiquitous enzyme, which is present at the protein and carbohydrate metabolism crossroads. The enzyme activity was investigated in biliary and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer, respectively, infecting the Indian water buffalo Bubalus bubalis. The enzyme activity was consistently higher in G. explanatum as compared to G. crumenifer, where NAD(H) was utilized as coenzyme and the pH optima was recorded at 8. The K(m) and V(max) values for α-ketoglutarate were 2.1 mM and 9.09 units in G. explanatum, whereas 3.03 mM and 1.90 units in G. crumenifer, respectively. Among the allosteric modulator nucleotides, AMP, ADP, ATP, GMP, CMP and UMP, only AMP enhanced GLDH activity in G. crumenifer while ADP was stimulatory in G. explanatum. The amino acid leucine stimulated the GLDH activity in both the amphistomes while alanine was stimulatory only in G. crumenifer. Pronounced interspecific differences in response to different metabolic inhibitors like diethyldithiocarbamate, semicarbazide hydrochloride and mercurial ions were also observed. The osmotic stress alters the enzyme activity, particularly in hypertonic saline the GLDH activity increased significantly (p < 0.01) in G. explanatum, while insignificant effects were observed in rumen dwelling G. crumenifer. Histoenzymology revealed region/tissue specific distribution of GLDH with prominent staining in tissues like vitellaria, lymph system and tegument/subtegument, thus showing specific distribution of GLDH indicating differential metabolic state. Such intergeneric differences in GLDH activity could also be a consequence of occupying different microenvironments within the same host.

Partial purification and characterization of Gigantocotyle explanatum somatic antigens.

Soluble extracts of Gigantocotyle explanatum, isolated from the liver of buffalo Bubalus bubalis were fractionated on Sephadex G-200 columns. Nine major fractions referred to as F1, F2, F3, F4, F5, F6, F7, F8 and F9 were separated. Each fraction was tested by ELISA for antigenicity using sera from G. explanatum-infected field buffaloes. Fractions F1 and F2 were highly antigenic, F3, F4, F6 and F7 were moderately antigenic and F5, F8 and F9 were poorly antigenic. Analyses by SDS-PAGE revealed that each fraction comprised several polypeptide(s) in the molecular weight range of <29 to >205 kDa. Results of Western blotting indicated that not all polypeptides which appeared in the SDS-PAGE were antigenic. The antigenic molecules of each fraction were mostly in the low molecular weight range of <14 to >94 kDa with the polypeptides in the range of >14, 14, 18, 21-25 and 34-36 kDa.

Analysis of potential antigens of protoscoleces isolated from pulmonary and hepatic hydatid cysts of Bubalus bubalis.

The aqueous soluble proteins of the protoscoleces of Echinococcus granulosus isolated from the pulmonary and hepatic hydatid cysts of Bubalus bubalis were partially purified on Sephadex G-200 column. The isolated fractions were tested for their antigenicity by immunodot using the sera collected from experimentally infected puppies during the prepatent period of infection. Six protein profiles (F1-F6) were recovered from both the isolates but the polypeptide recovery of each fraction of the two isolates were some what different, particularly protein concentration of F5 in the liver isolate was greater than the lung isolate. Contrary to this, the concentration of F1 polypeptides of the lung origin protoscoleces is greater as compared to that of the liver. In addition, some antigenic dissimilarity has also been observed in the two isolates. A weak IgG response against F1, F2 and F6 polypeptides was observed in the 4th day post infection (p.i.) sera. With the age of infection the response against these antigens increased as revealed by the intensity of the spot in immunodot analysis. Our studies show that the differences in the elution profile and antigenic profile of the lung and liver isolates, as revealed by gel filtration and immunodot analysis, might be either due to the influence of the microhabitat or these may be two different strains. Further studies are certainly required in this direction.

Partial purification and characterization of Gastrothylax crumenifer somatic antigens.

The soluble extracts of Gastrothylax crumenifer isolated from the rumen of buffalo (Bubalus bubalis) were fractionated on a Sephadex G-200 column. A total of eight major fractions (F1, F2, F3, F4, F5, F6, F7, and F8) were separated from the whole homogenate of G. crumenifer, and each of these fractions was tested for their antigenicity by ELISA against rabbit hyperimmune sera. It was observed that F1, F2, F3 and F4 were highly antigenic, F6 and F7 were moderately antigenic and F5 and F8 were poorly antigenic. The individual fractions analysed after SDS-PAGE and Western blotting indicated that the antigenic fractions of G. crumenifer are of low molecular weight, in the range of <14-50kDa, and predominant antigenic components which were evident in most of the Sephadex profiles were of Mr 15, 18, 19, 23-24 and 28-32kDa.

Analysis of excretory-secretory and somatic antigens of Gastrothylax crumenifer.

The excretory/secretory (ES) metabolic products released by Gastrothylax crumenifer (Trematoda: Digenea) during in vitro incubations and the somatic extract of the adult parasite were analysed using polyacrylamide gel electro-phoresis (PAGE). Immunogenicity of ES and somatic extracts were evaluated by immunoblotting and ELISA using sera raised against ES and somatic antigens in rabbits. The electropherograms of ES and somatic extracts have been resolved into 38 and 41 polypeptides, respectively. The apparent molecular weights of these polypeptides range from <29 to > 205 kDa. A total of 14 polypeptides were found to be common to both of the samples. The analysis of immunoblot results revealed 22 and 27 antigenic polypeptides in ES and somatic extracts respectively. Only 11 and 13 antigenic polypeptides were found specific to ES and tissue extract respectively. The molecular weights of these specific polypeptides were calculated to be <14.4, 16, 20, 25, 33, 42, 119, 125 and > 205 kDa for metabolic products and <14.4, 25, 30, 35, 78, 84 and > 205 kDa for the tissue extracts, respectively. Analysis of ELISA results revealed that a dilution of up to 1:3200 of the test sera could react with the ES product. Further, when the ES antigens were allowed to react with antisomatic extracts in hyperimmune sera the titre of IgG increased up to a dilution of 1:12800. The potential importance of these antigens in the immunodiagnosis of amphistomiasis is discussed.