Bioseparation and activity of Khaya senegalensis fractions against infective larvae of Haemonchus contortus.

The anthelmintic constituents of Khaya senegalensis (Deser.) A. Juss (Meliaceae) bark extract, previously demonstrated to show both in vitro and in vivo activity against gastrointestinal nematodes of sheep was investigated by chromatographic separation of the crude extract and bioassay of fractions. Bioseparation of the crude ethanol (95%) extract was carried out by gradient vacuum liquid chromatographic analysis and thin layer chromatography fingerprinting of eluates. The activity of the fractions was tested by a larval development assay in vitro. The survival rate of infective larvae L3 of sheep nematodes, predominantly Haemonchus contortus, was used to assess relative bioactivity. A secondary fraction obtained from further purification by preparative thin layer chromatography of a primary active fraction was also assessed for bioactivity. The compositions of the fractions were determined by qualitative chemical tests. The extracts killed infective larvae of H. contortus in a concentration-dependent manner. Best-fit LC50 values were 80.81, 63.73, 44.03 and 63.90 microg/ml for fractions A, B, C, and D, respectively (95% CI). The fractions are composed of saponins (A), saponins and alkaloids (B), saponins, terpenoids, flavonoids, condensed tannins (C), and saponins and tannins (D). Fraction C shows the highest activity of all the fractions, however, the difference is not statistically significant (p>0.05, Kruskal-Wallis test). The secondary fraction, C1A obtained from fraction C gave best-fit LC50 value of 5.09 microg/ml (95% CI) and was identified to be condensed tannin. The anthelminthic activity of K. senegalensis appears to involve synergism between various secondary metabolites found in the extract rather than a particular group of compounds.

Population dynamics of onchocerca volvulus microfilariae in human host after six years of drug control.

BACKGROUND & OBJECTIVES: Mass administration of ivermectin drug was carried out annually between 1995 and 2001 in three villages that were endemic for onchocerciasis in the Lower Cross River Basin, Nigeria. The aim of this study was to evaluate the population dynamics (dispersion patterns, distribution, prevalence and intensity) of Onchocerca volvulus microfilariae in their human host after six years of ivermectin treatment. METHODS: A total of 1014 subjects from three rural areas in Etung Local Government Area of Cross River State, Nigeria were screened for skin microfilariae using standard parasitological method of diagnosis. RESULTS: Ivermectin drug intervention had significantly reduced the prevalence of skin microfilariae (PMF) from 69.3% pre-control to 39.3% and community microfilarial load (CMFL) from 7.11 to 2.31 microfilariae per skin snip. Males (45%) were significantly (p <0.05) more infected than females (34%). Both microfilarial prevalence and intensity increased with age. Pearson correlation test between intensity and age was not significant (r = 0.37; p >0.05). The correlation between age-dependent parasite prevalence and mean abundance was also not significant (r = 0.42; p >0.05). The degree of dispersion as measured by variance to mean ratio (VMR), coefficient of variation (CV) and exponent 'K' of the negative binomial model of distribution showed that the parasite aggregated, clumped and overdispersed in their human host. The relative index of potential infection of each age group showed that adults between the age of 21 and 50 yr accounted for 52.7% of microfilariae positive cases. INTERPRETATION & CONCLUSION: Aggregated and overdispersion of O. volvulus observed in this study showed that active transmission could still be going on, because the tendency of the vector, Simulium damnosum ingesting more microfilariae was high due to the aggregated and overdispersed nature of the parasite with its host.

Anthelmintic activity of Spigelia anthelmia extract against gastrointestinal nematodes of sheep.

In vitro (larval development assay) and in vivo studies were conducted to determine possible direct anthelmintic effect of ethanolic and aqueous extracts of Spigelia anthelmia towards different ovine gastrointestinal nematodes. The effect of extracts on development and survival of infective larvae stage (L(3)) was assessed. Best-fit LC(50) values were computed by global model of non-linear regression curve fitting (95% confidence interval). Therapeutic efficacy of the ethanolic extracts administered orally at a dose rate of 125, 250, and 500 mg/kg, relative to a non-medicated control group of sheep harbouring naturally acquired infection of gastrointestinal nematodes, was evaluated in vivo.The presence of S. anthelmia extracts in the cultures decreased the survival of L(3) larvae. The LC(50) of aqueous extract (0.714 mg/ml) differ significantly from the LC(50) of the ethanolic extract (0.628 mg/ml) against the strongyles (p < 0.05, paired t-test). Faecal egg counts on day 12 after treatment showed that the extract is effective, relative to control (one-way analysis of variance [ANOVA], Dunnett's multiple comparison test) at 500 mg/kg against Strongyloides spp. (p < 0.01), 250 mg/kg against Oesophagostomum spp., Trichuris spp. (p < 0.05), and 125 mg/kg against Haemonchus spp. and Trichostrongylus spp. (p < 0.01). The effect of the doses is significant in all cases, the day after treatment is also extremely significant in most cases, whereas interaction between dose and day after treatment is significant (two-way ANOVA). S. anthelmia extract could, therefore, find application in the control of helminth in livestock, by the ethnoveterinary medicine approach.

Anthelmintic efficacy of Nauclea latifolia extract against gastrointestinal nematodes of sheep: in vitro and in vivo studies.

Direct effects of Nauclea latifolia extracts on different gastrointestinal nematodes of sheep is described. In vivo and in vitro studies were conducted to determine possible anthelmintic effect of leaf extracts of Nauclea latifolia toward different ovine gastro intestinal nematodes. A larval development assay was used to investigate in vitro, the effect of aqueous and ethanolic extracts of N. latifolia towards strongyles larvae. The development and survival of infective larvae (L(3)) was assessed and best-fit LC(50) values were computed by global model of non-linear regression analysis curve-fitting (95% CI). Twenty sheep harbouring naturally acquired gastrointestinal nematodes were treated with oral administration of ethanolic extracts at a dose rate of 125 mg/kg, 250 mg/kg and 500 mg/kg to evaluate therapeutic efficacy, in vivo.The presence of the extracts in the cultures decreased the survival of larvae. The LC(50) of aqueous and ethanolic extract were 0.704 and 0.650 mg/ml respectively and differ significantly (P<0.05, paired t test). Faecal egg counts (FEC) on day 12 after treatment showed that the extract is effective, relative to control (1-way ANOVA, Dunnett's multiple comparison test), at 500 mg/kg against Haemonchus spp, Trichostrongylus spp (p<0.05), Strongyloides spp (P < 0.01); at 250 mg/kg against Trichuris spp (P < 0.01) and ineffective against Oesophagostomum spp (p>0.05). The effect of doses is extremely significant; the day after treatment is sometimes significant while interaction between dose and day after treatment is insignificant (2-way ANOVA). N. latifolia extract could therefore find application in the control of helminth in livestock, by the ethnoveterinary medicine approach.

Physico-chemical indices of breeding sites of Simulium damnosum in the lower Cross River Basin, Nigeria.

To determine the factor influencing the abundance development and distribution of Simulium damnosum (S.I) immature stages in the lower Cross River Basin, Nigeria, the physico-chemical characteristics of water at breeding sites were analysed longitudinally over a 12 month period. Analysis of the water samples showed that water current correlated positively and significantly (p < 0.05), with dissolved oxygen (r = 0.95 vs 0.10); conductivity (r = 0.95 vs 0.81); hydrogen ion concentration (r = 0.03 vs 0.46); and biochemical oxygen demand (r = 0.80 vs 0.64); from Agbokim Waterfalls and Afi River respectively. The result indicates that these parameters and low ionic concentration are the most essential requirement for the development of S. damnosum pre-imaginal stages. Amplitudes of annual variability as measured by co-efficient of variation varied between the parameters. From the trends in hydrological variables it was deduced that the integrated impact of precipitation, input of surface runoff and municipal effluents played an overriding role in determining the absolute levels and temporal pattern in the water quality attributes. Of the 3578 pre-imaginal stages of S. damnosum collected, 52.52% and 47.4% were collected from Agbokim Waterfall and Afi River respectively. There was a significant difference (p < 0.05) in the number of pre-imaginal stages collected from both sites. The highest number of pre-imaginal stages of S. damnosum collected were at the peak of rain (July-September) indicating that S. damnosum in the River Basin is a wet season breeder. The need for long term laboratory colonization of blackfly immature stages becomes imperative especially how these parameters could be exploited in control programme through the testing of larvicides without adverse ecological damage to the aquatic habitat.

Anthelmintic activity of extracts of Spondias mombin against gastrointestinal nematodes of sheep: studies in vitro and in vivo.

This study was carried out to validate the efficacy of Spondias mombin, used locally as an anthelmintic, and to standardize the effective dose of the plant extract required for worm control in livestock. In vitro and in vivo studies were conducted to determine the direct anthelmintic effect of ethanolic and aqueous extracts of S. mombin towards different ovine gastrointestinal nematodes. A larval development assay (LDA) was used to investigate the in vitro effect of extracts on strongyle larvae. Another study was conducted in vivo to evaluate the therapeutic efficacy of the extracts administered orally at dose rates of 125, 250, 500 mg/kg to sheep naturally infected with gastrointestinal nematodes. Twenty sheep were selected on the basis of positive faecal egg counts (750 epg). The sheep were allocated randomly to a non-medicated control group (A) or to groups given 125 mg/kg (B), 250 mg/kg (C) or 500 mg/kg (D) of extract, respectively. Sheep in groups B-D were given extracts orally on two days. Individual faecal egg counts were performed on days 0, 3, 6, 9 and 12. The presence of S. mombin extracts in in vitro cultures of larvae decreased the survival of L3 larvae. The LC50 of the aqueous extract of S. mombin was 0.907 mg/ml, while the LC50 of the ethanolic extract was 0.456 mg/ml. This difference in LC50 was statistically significant (p > 0.05). The mean percentage faecal egg reduction of sheep drenched with 500 mg/kg S. mombin extracts was 15.0%, 27.5%, 65.0%, 65.0%, 100.0% against Haenmonchus spp., Trichostrongylus spp., Oesophagostomunm spp., Strongyloides spp. and Trichuris spp. respectively, on day 12. Extracts of S. mombin could find application in the control of helminths in livestock.

Evaluation of the anthelmintic activity of Khaya senegalensis extract against gastrointestinal nematodes of sheep: in vitro and in vivo studies.

The anthelmintic effect of Khaya senegalensis is described. In vitro and in vivo studies were conducted to determine possible direct anthelmintic effects of ethanolic and aqueous extracts of K. senegalensis towards different ovine gastrointestinal nematode. A larval development assay was used to investigate in vitro, the effect of aqueous and ethanolic extracts towards larvae of strongyles. The LC50 values of the effects of both the aqueous and ethanolic extracts were calculated. Another study was conducted in vivo to evaluate the therapeutic efficacy of the extracts administered orally at a dose rate of 125, 250 and 500 mg/kg of sheep harbouring naturally acquired infection of gastrointestinal nematodes. The presence of K. senegalensis extracts in the cultures decreased the viability of larvae. The LC50 of the aqueous extract (0.69 mg/ml) is not significantly different (P >0.05, t-test) from the ethanolic extract (0.51 mg/ml). The activity of the extract is concentration dependent in vivo. Sheep drenched with 500 mg/kg K. senegalensis ethanolic extract had a mean faecal egg count (FEC) reduction of 88.82%. The extract of K. senegalensis could find application in anthelmintic therapy in veterinary practice.

The use of monoclonal antibody for the immunodiagnosis of Fasciola gigantica infection in cattle.

Antigens that were specific to Fasciola gigantica were obtained from the whole worm homogenate of the parasite by immunoaffinity chromatography in cyanogen bromide-activated sepharose 4B columns and used for the production of monoclonal antibodies. The F. gigantica-specific monoclonal antibody was labelled with horseradish peroxidase and used for the detection of circulating antigen by the direct ELISA method in the sera of cattle experimentally infected with the parasite. Circulating antigens were detectable in the sera of the animals as from the third week after infection while negative absorbance values were obtained 2 weeks after the termination of the infection by chemotherapy with oxyclozanide. This immunodiagnostic method offers an attractive alternative as a supplement to the conventional coprological diagnosis of fasciolosis.

The isolation of Fasciola gigantica-specific antigens and their use in the serodiagnosis of fasciolosis in sheep by the detection of circulating antigens.

The 17kDa and 69kDa polypeptide antigens which are specific to Fasciola gigantica were excised from polyacrylamide gels and used for the immunization of rabbits to raise monospecific antisera against these polypeptides. These sera were labelled with horseradish peroxidase and used for the detection of circulating 17 kDa and 69 kDa antigens by a direct enzyme-linked immunosorbent assay in the sera of sheep that were experimentally or naturally infected with F. gigantica. The 17 kDa antigen was detected in the sera of infected sheep as early as 1 week after infection and, following chemotherapy with oxyclozanide, negative seroconversion occurred 2 weeks later. The 69 kDa antigen was detectable as from the fourth week of infection and its detection ceased 3 weeks after chemotherapy. The serodiagnosis of F. gigantica, based on the detection of the 17 kDa antigen in sheep sera, was more specific and sensitive than that based on the detection of the 69 kDa antigen.

Prevalence and seasonal changes in the gastro-intestinal helminths of Nigerian goats.

A total of 120 gastro-intestinal tracts and 960 faecal samples were examined to assess the prevalence and seasonal changes in the gastro-intestinal helminth parasites of Red Sokoto (maradi) goats slaughtered at Ibadan between May 1991 and April 1992. Egg types of strongyles, Strongyloides, Trichuris, Skrjabinema, Dicrocoelium and Moniezia were encountered in 93%, 83%, 44%, 0.9%, 2.3% and 31% of the faecal samples respectively. However, only strongyle, Strongyloides and Trichuris eggs occurred in large numbers and were more common during the rainy season than in the dry season. The parasites recorded and their prevalences were Haemonchus contortus (90.0%), H. ovis (5.0%), Strongyloides papillosus (80.8%), Trichostrongylus colubriformis (78.3%), T. axei (69.2%), Trichuris ovis (72.5%), T. globulosa (38.3%), Oesophagostomum columbianum (67.5%), Cooperia curticei (58.3%) Gaigeria pachyscelis (40.8%), Skrjabinema ovis (5.0%), Nematodirus battus (5.8%), Moniezia expansa (29.2%), M. benedeni (10.0%), Paramphistomum spp. (5.0%) and Cysticercus tenuicollis (33.3%). Haemonchus ovis is reported for the first time in Nigeria. Mixed infections were most prevalent. Young goats were more commonly infected and had higher worm counts than adult goats. Only Haemonchus, Trichostrongylus, Strongyloides and Cooperia spp. occurred in large numbers. Irrespective of the age of the goats, higher worm counts were generally encountered during the rainy season than in the dry season. The results are discussed in relation to the control of helminthiasis in grazing animals in Nigeria.

The immune response of Trypanosoma gambiense-infected mice to Schistosoma whole worm antigens.

Heterologous humoral and cellular immune response to Schistosoma mansoni and S. bovis antigens were investigated in Tryponosoma gambiense-infected mice in order to understand the concurrent immune response to both parasites. Immune response as measured by the Indirect Haemagglutination Test (IHA), Spontaneous sheep red blood cell rosette formation with B-lymphocytes and the mean number of T-lymphocytes forming rosettes with AET-treated SRBC to the schistosome antigens, was significantly depressed in the trypanosome-infected mice. Furthermore, the timing of trypanosome infection and the inoculation of antigen was related to the degree of immunosuppression as animals which had trypanosome infection and the whole worm antigen of S. mansoni and S. bovis simultaneously had higher IHA titre than those which were inoculated with the antigens 10 days after the commencement of an infection. The results are discussed from the viewpoint of immunological control of schistosomiasis in areas where both diseases are endemic.

Antibody and circulating antigen profiles before and after chemotherapy in goats infected with Fasciola gigantica.

The profiles of antibody response and circulating antigen levels in goats infected with Fasciola gigantica were studied by enzyme-linked immunoelectrotransfer blot (EITB) and enzyme-linked immunosorbent assay (ELISA). In the antibody assay, sera from goats experimentally infected with F. gigantica were reacted with whole worm antigen of the worm before and after chemotherapy with oxyclozanide. In ELISA, there was a significant increase in antibody level 2 weeks after infection. After chemotherapy, there was a gradual decrease in antibody within 3 weeks followed by a rapid decline by the 4th week after treatment. By EITB, the infected goat sera recognized three polypeptides in the range of 42-80 kDa as early as 2 weeks after infection. Recognition of the complete components of F. gigantica antigen repertoire occurred as early as the 4th week after infection. By the 8th week after chemotherapy, distinct polypeptide band recognition was no longer possible. Comparative immunoblotting with goat anti-Paramphistomum, anti-Dicrocoelium and anti-Fasciola sera revealed that the 14 kDa, 17 kDa, 21 kDa, 28 kDa and 30 kDa proteins are specific to F. gigantica. In the antigen assay, circulating antigen was detectable by the direct ELISA method one week after infection and negative absorbance values were observed 4 weeks after chemotherapy.

Detection of circulating Fasciola gigantica antigen in experimental and natural infections of sheep with fasciolosis.

Detection of circulating Fasciola gigantica antigen was performed in sera of sheep with experimental and natural F. gigantica infections using the direct enzyme-linked immunosorbent assay method. Sera from sheep with monoinfections of Schistosoma bovis, Dicrocoelium hospes and Paramphistomum microbothrium were included in the assay to ascertain specificity. Circulating F. gigantica antigen (CFA) was detected as early as 1 week after infection in the experimentally infected sheep. No detectable CFA was observed 2 weeks after chemotherapy. Positivity rates of 82.5%, 12.5%, 10% and 10% were found in sera with monospecific infections of F. gigantica, P. microbrothrium, D. hospes and S. bovis, respectively. Acid treatment of the sera did not enhance the sensitivity of the assay.

Time-course analysis of antibody response by EITB and ELISA before and after chemotherapy in sheep infected with Fasciola gigantica.

The time-course analysis of the antibody response to Fascicola gigantica infection in sheep was studied by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunoelectrotransfer blot (EITB). Sera from sheep experimentally infected with F. gigantica were reacted with excretory-secretory antigens of the worm before and after chemotherapy with oxyclozanide. In ELISA, there was a significant increase in anti-Fasciola antibody by 2 weeks after infection and there was a sharp decrease in antibody titer by 4 weeks after treatment. By EITB, the infected sheep sera recognised four polypeptides in the range of 43-75 kDa as early as 2 weeks after infection, with more polypeptides being recognised as the infection progressed. Recognition of an 87 kDa antigen was lost by 2 weeks after treatment and is therefore a good marker for treatment efficacy. Comparative immunoblotting with sheep anti-Paramphistomum, anti-Dicrocoelium and anti-Fasciola sera revealed that the 17 kDa, 21 kDa, 57 kDa and 69 kDa proteins are specific to fasciolosis and are good antigens for early and specific immunodiagnosis of F. gigantica infection in sheep.

Detection of circulating antigen in sera of Fasciola gigantica-infected cattle with antibodies reactive with a Fasciola-specific 88-kDa antigen.

Antibodies against a specific 88-kDa antigen of Fasciola gigantica were used for the detection of circulating antigen in the sera of cattle with experimental and natural infections of F. gigantica by a double antibody ELISA. Circulating antigen was detectable as early as the second and third weeks after infection and positive absorbance values were obtained for the entire duration of infection. Absorbance values decreased below the cutoff point 3 weeks after chemotherapy with oxyclozanide. This immunoassay also greatly enhanced the specificity of immunodiagnosis of fasciolosis in naturally infected cattle. The test system has excellent potential for the accurate diagnosis of ruminant fasciolosis.

Immunodiagnosis of fasciolosis in ruminants using a 28-kDa cysteine protease of Fasciola gigantica adult worms.

A homogeneous extract of a 28-kDa cysteine protease of Fasciola gigantica adult worms was used as the antigen for immunodiagnosis of fasciolosis in cattle, sheep and goats using the Falcon assay screening test enzyme-linked immunosorbent assay technique. This antiprotease assay technique was found to be very rapid and sensitive and may be useful as a supplementary method for the diagnosis of fasciolosis in ruminants.

Development and characterization of a monoclonal antibody reactive with a 28 kDa protease of Fasciola gigantica.

A monoclonal antibody that was reactive with a 28-kDa protease of Fasciola gigantica was produced after hyperimmunizing BALB/c mice with a partially purified extract and a purified 28-kDa protease obtained from the fluke. Isotype analysis showed that the immunoglobulin is IgG3. Seven other monoclonals of isotypes IgG1, IgG2 and IgG3 which reacted with the protease and one to three other protein bands were also produced. In addition, four other clones of hybridoma produced antibodies which recognised a 27/28-kDa protein doublet but the 28-kDa component of this doublet was different from the 28-kDa protease. The reactivities and characteristics of the monoclonal antibodies were studied by enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunoprecipitation tests.

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

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