Survival of anopheline eggs and their susceptibility to infection with Metarhizium anisopliae and Beauveria bassiana under laboratory conditions

The viability of Anopheles gambiae sensu stricto and Anopheles arabiensis (Diptera: Culicidae) eggs over time and the ovicidal activity of Beauveria bassiana (Ascomycota: Cordycipitaceae) and Metarhizium anisopliae (Ascomycota: Clavicipitaceae) were investigated. Eggs were incubated in soil or leaf litter for up to 12 weeks at 26°C and 75%, 86% or >98% relative humidity (RH). Eggs were treated topically with M. anisopliae ICIPE-30 or B. bassiana I93-825 conidia in either water or oil-in-water formulations. Survival of eggs whether treated or not with fungus was similar, and untreated eggs generally did not survive longer than 2 weeks regardless of the substrate or humidity tested. After a minimal 5-day exposure, M. anisopliae at 5 × 10⁶ conidia/cm² clearly reduced the number of larvae. The efficacy of the fungus increased when it was oil-in-water formulated, and eclosion was completely prevented regardless of the conidial concentration (10⁵–10⁷ conidia/cm²) after a 10-day exposure in soils at >98% RH. Treatment of eggs with B. bassiana, however, failed to reduce the number of eclosing larvae. This is the first demonstration of the ovicidal activity by M. anisopliae against either A. gambiae s. s. or A. arabiensis and the results underline the potential of this fungus against anopheline mosquitoes.     

Adulticidal and larvicidal activity of Beauveria bassiana and Metarhizium anisopliae against housefly, Musca domestica (Diptera: Muscidae), in laboratory and simulated field bioassays

The susceptibility of the adult and larval stage of housefly, Musca domestica L. (Diptera: Muscidae), to two entomopathogenic fungi, Metarhizium anisopliae (Metsch.) Sor. and Beauveria bassiana (Bals.) Vuill., was evaluated under laboratory and simulated field bioassays. Bioassays on adult houseflies were carried out at different conidial concentrations ranging from 10³ to 10⁹ conidia/ml in petri plate and minichamber assays. Absolute mortality was observed within 4–5 days at all the concentrations tested. M. anisopliae was found to be more effective with LC₅₀ of 6.75 × 10⁷ conidia/ml compared with 1.21 × 10⁸ conidia/ml of B. bassiana in petri plate bioassay. Similar trend was observed in minichamber bioassay. Larvicidal activity evaluated through petri plate bioassay also indicated that M. anisopliae was more effective larvicide with LC₅₀ of 4.1 × 10⁸ conidia/ml as against 3.31 × 10⁹ conidia/ml of B. bassiana. Larvicidal activity was further evaluated in simulated field condition of decaying waste matrix using dry conidial formulations (10⁸ conidia/g) of both the fungi. Larval mortality obtained in this assay was 43% (B. bassiana) and 63% (M. anisopliae). Remarkably better performance of M. anisopliae as an adulticidal and larvicidal agent over B. bassiana in laboratory bioassays as well as simulated field conditions suggests that it may have good potential to become part of an integrated housefly control program.     

Laboratory and field evaluation of the fungus Chrysosporium lobatum against the larvae of the mosquito Culex quinquefasciatus

Eleven fungal species in three genera were isolated from the soil at Agra, India by the feather-baiting technique. Out of the 11 species, Chrysosporium lobatum Scharapov, a deuteromycetous (Moniliales: Moniliaceae) fungus, caused high mortality of Culex quinquefasciatus Say (Diptera: Culicidae) larvae in the laboratory. Laboratory and field trials were carried out to study the efficacy of C. lobatum against various instars of C. quinquefasciatus. In the laboratory bioassays, first, second and third instar larvae were assessed separately. Conidia of C. lobatum that were 15 days old were used both in laboratory and field bioassays. Six different concentrations were used in the laboratory bioassays (10, 10³, 10⁴, 5 × 10⁴, 10⁵ and 10⁶ conidia/mL), and one concentration was tested in the field trials (10⁶ conidia/mL). The LC₅₀ values with 95% fiducial limits and probit equations were calculated by the probit analysis. Significant (analysis o.f variance (ANOVA): P < 0.0001) mortality difference between the instars of C. quinquefasciatus was observed after 72 h of exposure to conidia of C. lobatum. The third instar C. quinquefasciatus were 319- and 25-fold more susceptible to C. lobatum (0.47 × 10³ conidia/mL) than the first and second instars, respectively. The cuticle of the abdominal region and anal papillae were densely covered by the C. lobatum conidia. In the field trials, populations had declined significantly (t test, P = 0.003) 15 days after inoculation in the four test pools. Significantly (t test, P = 0.03) higher mortality was observed in the pools with water quality that was lower in total dissolved solids, hardness, chemical oxygen demand and conductivity. Based on the performance and survival in the trial, C. lobatum may be considered as a bio-control agent of mosquitoes.     

Effect of praziquantel prolonged administration on granuloma formation around Schistosoma japonicum eggs in lung of sensitized mice

Schistosomiasis remains a major public health problem and it is an immune disease. The schistosome egg is the primary parasite factor responsible for the overt disease. The eggs release the soluble antigen, which induces intensive tissue reaction, a granulomatous reaction to the eggs. If granuloma formation could be suppressed, overt disease might not develop. Praziquantel is an effective antischistosomal drug especially for adult worms. However, whether praziquantel has a suppressing effect on granuloma formation around schistosome eggs directly remains unclear. The purpose of the present study was to investigate the effect of praziquantel, especially administered persistently, on granuloma formation around Schistosoma japonicum eggs in the lung of sensitized mice. Thirty-six mice were divided into three groups averagely. Group A was a control group. First, the mice were injected with schistosomal eggs hypodermically in abdomen, and 10 days later injected with schistosomal eggs intravenously via a tail vein. Group B was a praziquantel short administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 300 mg/kg for 3 days, from 1 day before the intravenous injection of the eggs. Group C was a praziquantel prolonged administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 150 mg/kg for 5 days weekly until the mice were sacrificed. Three mice of each group were sacrificed on days 7, 14, 28, and 56, respectively after the intravenous injection of the eggs, and the lung tissues were fixed with formalin and the slices were HE stained. The granulomas containing eggs in their centers were selected, and 25–30 granulomas from the animals of each group were measured at each time period. The mean areas of egg granulomas of each group were calculated, and the neutrophilic granulocytes, eosinocytes, lymphocytes, fibroblasts, and macrophages within the egg granulomas were counted. The mean numbers of them of each group were calculated. All the data of each group were analyzed and compared statistically. On day 56 after the intravenous injection of the eggs, the mean area of schistosomal egg granulomas in group B was (227.4 ± 728.0) × 10³ μm², less than that of [(297.9 ± 153.3) × 10³ μm²] in group A, and the suppression rate was 23.7% (P < 0.05). On days 7, 14, 28, and 56, the mean areas of schistosomal egg granulomas in group C were (575.8 ± 155.6) × 10³ μm², (310.5 ± 854.0) × 10³ μm², (267.7 ± 513.3) × 10³ μm², and (214.9 ± 446.4) × 10³ μm², respectively, significantly less than those of [(692.7 ± 232.6) × 10³ μm², (439.4 ± 165.0) × 10³ μm², (385.7 ± 129.3) × 10³ μm², and (297.9 ± 153.3) × 10³ μm²] in group A. The suppression rates were 16.9%, 29.3%, 30.6%, and 27.9%, respectively (P values <0.05). On day 56, the mean numbers of neutrophilic granulocytes were 11.4 ± 5.0 in group A and 5.2 ± 3.1 in group C, respectively, with the suppression rate of 54.4% in group C (P < 0.05). On day 56, the mean numbers of eosinocytes within the egg granulomas were 2.3 ± 2.0, 0.1 ± 0.3, and 0.3 ± 0.6 in groups A, B, and C, respectively, with the suppression rate of 95.7% in group B and 87.0% in group C (P values <0.05). On day 56, the mean numbers of macrophages within egg granulomas were 14.3 ± 6.9 in group C, compared with 18.6 ± 8.2 in group A, the suppression rate was 23.1% (P < 0.05). On day 56, the mean numbers of fibroblasts within the egg granulomas were 6.6 ± 4.4 and 5.8 ± 2.6 in groups B and C, respectively, and compared with 14.3 ± 7.8 in group A, the increasing extents decreased by 53.8% and 59.4%, respectively (P values <0.05). Therefore, the administration of praziquantel, especially the prolonged administration, can suppress the formation of schistosomal egg granulomas, including reduction in the areas of granulomas and suppression of the inflammatory cells and the hyperplasia of fibroblasts within granulomas.     

Insecticidal, acaricidal and repellent effects of DEET- and IR3535-impregnated bed nets using a novel long-lasting polymer-coating technique

A novel long-lasting repellent-treated net (LLRTN) has been designed by binding the skin repellents N,N-diethyl-m-toluamide (DEET), or IR3535, onto the fibres of bed net fabric using a new polymer-coating technique. The repellent toxicological effectiveness and residual activity of a factory-based repellent-impregnated fabric has been evaluated by laboratory testing against adult Aedes aegypti mosquitoes and nymphal Ixodes ricinus ticks. By using this repellent-embedding impregnation technique, concentrations exceeding 10 g/m² could be achieved with one single polymer layer. Both DEET- and IR3535-impregnated fabrics revealed a dose-dependent insecticidal as well as acaricidal activity. One hundred percent knockdown times of DEET-treated bed nets ranged from 187.5 ± 31.8 to 27.5 ± 3.5 min against A. aegypti, and between 214 ± 47 and 22.6 ± 5 min against nymphal I. ricinus, linked to a DEET concentration of 1.08 and 10.58 g/m², respectively. With IR3535, A. aegypti produced dose-dependent 100% knockdown times varying from 87.5 ± 10.6 to 57.5 ± 3.5 min and between 131.4 ± 6.5 and 33.8 ± 5 min against nymphal I. ricinus, respectively, linked to concentrations between 1.59 and 10.02 g/m². One hundred percent repellency measured by complete landing and biting protection of impregnated fabric by using the arm-in-cage test could be achieved at DEET concentrations exceeding 3.7 to 3.9 g/m², and for IR3535 concentrations over 10 g/m². One hundred percent landing and biting protection could be preserved with DEET-treated fabrics for 29 weeks at an initial concentration of 4.66 g/m², 54 weeks at 8.8 g/m², 58 weeks at 9.96 g/m² and 61 weeks at 10.48 g/m² for DEET, and 23 weeks for IR3535-treated fabric at a concentration of 10.02 g/m². Unlike repellent-treated fabric, a brand of a commercially available long-lasting insecticide-treated net tested containing 500 mg permethrin/m² did not protect from mosquito bites. First results on bioactivity and long-lasting efficacy show that the new LLRTN technique is highly promising as a potential candidate for future malaria control strategies, especially in areas where pyrethroid resistance occurs.     

Ovicidal effects of a neem seed extract preparation on eggs of body and head lice

The eggs (nits) of head and body lice (Pediculus humanus capitis, Pediculus humanus corporis) were incubated for 5, 10, 15, 20, 30 or 45 min into a neem seed extract contained in a fine shampoo formulation (e.g. Wash Away? Louse), which is known for its significant killing effects of larvae and adults of head lice. The aim of the study was to test whether the developmental stages inside the eggs are also killed after the incubation into the shampoo. It was found that an incubation time of only 5 min was sufficient to prohibit any hatching of larvae, whilst 93 ± 4% of the larvae in the untreated controls of body lice hatched respectively about 76% of the controls in the case of head lice. Apparently, the neem-based shampoo blocked the aeropyles of the eggs, thus preventing the embryos of both races of lice from accessing oxygen and from releasing carbon dioxide. Thus, this product offers a complete cure from head lice upon a single treatment, if the lice (motile stages, eggs) are fully covered for about 10 min.     

Infection of Anopheles gambiae mosquitoes with entomopathogenic fungi: effect of host age and blood-feeding status

Physiological characteristics of insects can influence their susceptibility to fungal infection of which age and nutritional status are among the most important. An understanding of host–pathogen interaction with respect to these physiological characteristics of the host is essential if we are to develop fungal formulations capable of reducing malaria transmission under field conditions. Here, two independent bioassays were conducted to study the effect of age and blood-feeding status on fungal infection and survival of Anopheles gambiae s.s. Giles. Mosquitoes were exposed to 2 × 10¹⁰ conidia m⁻² of oil-formulated Metarhizium anisopliae ICIPE-30 and of Beauveria bassiana I93-825, respectively, and their survival was monitored daily. Three age groups of mosquitoes were exposed, 2–4, 5–8, and 9–12 days since emergence. Five groups of different feeding status were exposed: non-blood-fed, 3, 12, 36, and 72 h post-blood feeding. Fungal infection reduced the survival of mosquitoes regardless of their age and blood-feeding status. Although older mosquitoes died relatively earlier than younger ones, age did not tend to affect mosquito susceptibility to fungal infection. Non-blood-fed mosquitoes were more susceptible to fungus infection compared to all categories of blood-fed mosquitoes, except for those exposed to B. bassiana 72 h post-blood feeding. In conclusion, formulations of M. anisopliae and B. bassiana can equally affect mosquitoes of different age classes, with them being relatively more susceptible to fungus infection when non-blood-fed.     

Haematology and serum biochemistry of golden eagle (Aquila chrysaetos) in Iran

Haematological and serum biochemical values were estimated in blood samples collected from 21 apparently adult golden eagles (Aquila chrysaetos) of both sexes. The mean values of red blood cells, packed cell volume, haemoglobin, white blood cells, heterophils, lymphocytes, monocytes and eosinophils were 1.63 ± 0.11 × 10¹²/l, 0.47 ± 0.009 l/l, 91.73 ± 1.52 g/l, 24.31 ± 1.97 × 10⁹/l, 4.40 ± 0.22 × 10⁹/l, 16.81 ± 0.65 × 10⁹/l, 0.99 ± 0.19 × 10⁹/l and 2.10 ± 0.30 × 10⁹/l, respectively. The leucocytes had 69.14%, 4.09%, 18.12% and 8.65% lymphocytes, monocytes, heterophils and eosinophils, respectively. The results of serum biochemistry in the golden eagle indicated that the concentrations of glucose, total protein, albumin, total globulin, cholesterol, triglyceride, uric acid, blood urea nitrogen, creatinine, calcium, phosphorous, aspartate aminotransferase, alanine aminotransferase, creatine kinase, lactate dehydrogenase and alkaline phosphatase were 16.42 ± 0.73 mmol/l, 49.76 ± 1.35 g/l, 20.46 ± 0.79 g/l, 29.30 ± 1.47 g/l, 2.14 ± 0.09 mmol/l, 2.04 ± 0.08 mmol/l, 457.67 ± 97.46 μmol/l, 2.74 ± 0.17 mmol/l, 53.27 ± 3.87 μmol/l, 2.37 ± 0.24 mmol/l, 1.73 ± 0.08 mmol/l, 293.24 ± 18.96 IU/l, 28.21 ± 2.36 IU/l, 411.29 ± 58.37 IU/l, 1,209.89 ± 21.73 IU/l and 67.31 ± 5.29 IU/l, respectively. There were no significant differences between haematological and serum biochemical parameters of male and female golden eagles (P > 0.05).     

Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis

The sensitivity of LAMP, PCR and in vitro culture methods for the detection of Theileria equi and Babesia caballi was evaluated using tenfold serially diluted culture parasites. On day 1 post-culture, both T. equi and B. caballi parasites could only be observed at 1% parasite dilution from the in vitro culture method, whereas LAMP could detect up to 1 × 10⁻³% of both T. equi and B. caballi parasite dilutions, whilst PCR could detect 1 × 10⁻³% T. equi and 1 × 10⁻¹% B. caballi parasite dilutions. On day 7 post-culture, the detection limit for T. equi and B. caballi in the in vitro culture increased up to 1 × 10⁻⁶%, whereas LAMP detection limit increased to 1 × 10⁻¹⁰% for both parasites, whilst the PCR detection limit increased to 1 × 10⁻¹⁰% and 1 × 10⁻⁶% for T. equi and B. caballi, respectively. Furthermore, LAMP and PCR amplified the T. equi DNA extracted from the organs of an experimentally infected horse. This study further validates LAMP as an alternative molecular diagnostic tool, which can be used in the diagnosis of early infections of equine piroplasmosis and together with PCR can also be used as supplementary methods during post-mortems.     

Validation of models to estimate the fumigant and larvicidal activity of Eucalyptus essential oils against Aedes aegypti (Diptera: Culicidae)

The aim of this work is to validate the pre-existing models that relate the larvicidal and adulticidal activities of the Eucalyptus essential oils on Aedes aegypti. Previous works at our laboratory described that the larvicidal activity of Eucalyptus essential oils can be estimated from the relative concentration of two main components (p-cymene and 1,8-cineole) and that the adulticidal effectiveness can be explained, to a great extent, by the presence of large amounts of the component 1,8-cineole in it. In general, the results show that the higher adulticidal effect of essential oils the lower their larvicidal activity. Fresh leaves was harvested and distilled. Once the essential oil was obtained, the chemical composition was analysed, evaluating the biological activity of 15 species of the genus Eucalyptus (Eucalyptus badjensis Beuzev and Welch, Eucalyptus badjensis × nitens, Eucalyptus benthamii var Benthamii Maiden and Cambage, Eucalyptus benthamii var dorrigoensis Maiden and Cambage, Eucalyptus botryoides Smith, Eucalyptus dalrympleana Maiden, Eucalyptus fastigata Deane and Maiden, Eucalyptus nobilis L.A.S. Johnson and K.D.Hill, Eucalyptus polybractea R. Baker, Eucalyptus radiata ssp radiata Sieber ex Spreng, Eucalyptus resinifera Smith, Eucalyptus robertsonii Blakely, Eucalyptus robusta Smith, Eucalyptus rubida Deane and Maiden, Eucalyptus smithii R. Baker). Essential oils of these plant species were used for the validation of equations from preexistent models, in which observed and estimated values of the biological activity were compared. The regression analysis showed a strong validation of the models, re-stating the trends previously observed. The models were expressed as follows: A, fumigant activity [KT_50(min) = 10.65–0.076 × 1,8-cineole (%)](p < 0.01; F, 397; R ², 0.79); B, larval mortality (%)_(40 ppm) = 103.85 + 0.482 × p-cymene (%) − 0.363 × α-pinene (%) − 1.07 × 1,8-cineole (%) (p < 0.01; F, 300; R ², 0.90). These results confirmed the importance of the mayor components in the biological activity of Eucalyptus essential oils on A. aegypti. However, it is worth mentioning that two or three species differ in the data estimated by the models, and these biological activity results coincide with the presence of minor differential components in the essential oils. According to what was previously mentioned, it can be inferred that the model is able to estimate very closely the biological activity of essential oils of Eucalyptus on A. aegypti.     

Biological control of Rhipicephalus (Boophilus) annulatus by different strains of Metarhizium anisopliae, Beauveria bassiana and Lecanicillium psalliotae fungi

Virulence of 11 native strains of entomopathogenic fungi; Metarhizium anisopliae (three strains), Beauveria bassiana (six strains) and Lecanicillium psalliotae (two strains) collected from different parts of Iran, were studied against different developmental stages of Rhipicephalus (Boophilus) annulatus. After the exposure of ticks to the fungal strains in different concentrations (i.e. 10³, 10⁵, 10⁷ conidia/ml), various parameters such as mortality rate and reproductive efficiency of engorged females, mortality of unfed tick larvae and eclosion percentage of infected eggs were evaluated to determine the fungal virulence. Based on the obtained results, five strains including M. anisopliae (IRAN 437 C and DEMI 001), B. bassiana (IRAN 403 C) and L. psalliotae (IRAN 468 C and IRAN 518 C) were found to be virulent to various stages of tick developmental cycle. Mortality rate of engorged females was found to be dose-dependent with regard to the conidial concentration used. Total mortality rates of 90–100%, 70% and 56.6% were observed for M. anisopliae (IRAN 437 C and DEMI 001), B. bassiana (IRAN 403 C) and L. psalliotae (IRAN 468 C), 6–11 days post inoculation (PI) with 10⁷ conidia/ml, respectively. Most strains were able to inhibit egg laying by females in the range of 0–26% in different conidial concentrations. The results indicated that the mean egg laying of treated engorged tick females exposed to M. anisopliae (IRAN 437 C) was less than the mean values of those treated with other fungal strains. Results revealed 89.1%, 35.5% and 56.3% decrease in egg hatchability and 88.69%, 78.15% and 59.74% reduction in reproductive efficiency of the ticks using 10⁷ conidia/ml of M. anisopliae (IRAN 437 C), B. bassiana (IRAN 403 C) and L. psalliotae (IRAN 468 C), respectively. In general, the entomopathogenic effects of native M. anisopliae and B. bassiana against various developmental stages of R. (B.) annulatus were confirmed in the present work. Likewise, although L. psalliotae, which was introduced for the first time as an entomopathogenic fungus against tick had not more than 13.3% mortality effect against adult females, but its effect on egg hatchability and reproductive efficiency was remarkable.     

Echinococcus granulosus: membrane permeability of secondary hydatid cysts to albendazole sulfoxide

The objectives of the present study were, first, to establish a methodology for evaluation of the permeability in vitro of hydatid cysts to different drugs and, second, to compare the permeability to albendazole sulfoxide of cysts from untreated animals, cysts from animals treated with 50 mg/kg netobimin for 5 days, and cysts from animals treated with 50 mg/kg netobimin plus 1.1 mg/kg fenbendazole for 5 days. The drug flow follows the Fick law, i.e., the uptake occurs by simple diffusion. We calculated the permeability constant of the cyst membrane by taking into account the disappearance velocity constant, the cyst area, and the incubation solution volume. The permeability value obtained for albendazole sulfoxide was 8.06 ± 2.30 × 10⁻⁶ cm s⁻¹ in cysts from untreated animals, 5.56 ± 2.53 × 10⁻⁶ cm s⁻¹ in cysts from animals treated with netobimin, and 7.05 ± 3.04 × 10⁻⁶ cm s⁻¹ in cysts from animals treated with netobimin + fenbendazole. These permeability values show significant differences (P < 0.05). Received: 20 September 1997 / Accepted: 15 October 1997     

Determination of the effect of probiotic (Saccharomyces cerevisiae) on growth performance and hematological parameters of rabbits

Insufficient supply of animal protein is a major problem in developing countries including Nigeria. Rabbits are adjudged to be a convenient source of palatable and nutritious meat, high in protein, and contain low fat and cholesterol. A doe can produce more than 15 times her own weight in offspring in a year. However, its productivity may be limited by inadequate nutrition. The objective of this study was to determine the effect of probiotic (Saccharomyces cerevisiae) supplementation on growth performance and some hematological parameters of rabbit. The appropriate level of the probiotic inclusion for excellent health status and optimum productivity was also determined. A total of 40 male rabbits were randomly divided into four groups (A–D) of ten rabbits each. Each group was subdivided into two replicates of five rabbits each. They were fed pelleted grower mash ad libitum. The feed for groups A to C were supplemented with bioactive yeast (probiotic) at inclusion levels of 0.08, 0.12, and 0.16 g yeast/kg diet, respectively. Group D had no yeast (control). Daily feed intake was determined. The rabbits were weighed weekly. The packed cell volume (PCV), hemoglobin concentration, white blood cell total, and differential counts were determined at the 8th week, 16th week, and 22nd week following standard procedures. The three results which did not have any significant difference were pooled together. Group A which had 0.08 g yeast/kg of diet had a significantly lower (P ≤ 0.05) PCV than groups B (which had 0.12 g yeast/kg of diet) and C (which had 0.16 g yeast/kg of diet) as well as D (the control). Total WBC count for groups B and C (14.35 ± 0.100 × 10³/μl and 14.65 ± 0.786 × 10³/μl, respectively) were significantly higher (P ≤ 0.05) than groups A and D (6.33 ± 0.335 × 10³/μl and 10.40 ± 0.296 × 10³/μl, respectively). Also the absolute neutrophils and lymphocytes counts were significantly higher (P ≤ 0.05) in groups B and C than in groups A and D. Group B had significantly higher (P ≤ 0.05) weight gain (1.025 ± 0.006 kg/rabbit) followed by group A (0.950 ± 0.092 kg/rabbit). The control (group D) had the least weight gain of 0.623 ± 0.0.099 kg/rabbit. These results showed that like most probiotics, bioactive yeast at an appropriate level of inclusion had a significant beneficial effect on health status and growth rate of rabbit. Probiotic supplementation level of 0.12 g yeast/kg of diet was recommended for optimum rabbit production.     

Cryptosporidium parvum oocysts in zebra mussels (Dreissena polymorpha): evidence from the St. Lawrence River

Molluscan shellfish can recover and concentrate environmentally derived waterborne pathogens and can be used for the sanitary assessment of water quality. Oocysts of Cryptosporidium parvum (genotype 1) were identified in zebra mussels (Dreissena polymorpha) from the St. Lawrence River, Quebec. Approximately 67 oocysts/ml of hemolymph and 129 oocysts/g of soft tissue were recovered. The adjusted concentration of oocysts per gram of tissue was 2.2 × 10², and approximately 4.4 × 10² oocysts were recovered from a single mussel. Zebra mussels can serve as biological indicators of waterborne contamination with Cryptosporidium. Received: 22 May 2000 / Accepted: 17 July 2000     

Description and development of the early third-stage larva of Gnathostoma turgidum Stossich, 1902 (Nematoda: Gnathostomatidae) and contributions to its life cycle

The egg and larval stages of Gnathostoma turgidum were examined using light microscopy. Fertilized uterine eggs are 65.97 long and 32.28 wide, oval, brownish, with two cap-like thickenings. The eggshell surface is covered with numerous irregularly shaped pits of various sizes and depths. A sheathed second-stage larva emerges from the egg, measures 178 × 9; the sheath measures 243 × 21. Development to early third-stage larva in the coelomic cavity of cyclopoid copepods is similar to that described for other gnathostome species. After 10 days at 27°C, the larvae undergo a molt (the second for gnathostomes) and develop to early third stage. The body of this stage measures 412.3 × 40.1, with evident hemispherical cephalic bulbs. Cephalic bulbs measure 25 × 40, armed with four transverse rows of sharp hooklets. The average number of hooklets in each row is 31, 34, 37, and 42, respectively. The whole body is covered with 193 transverse rows of small single-pointed cuticular spines. One pair of cervical papillae and an excretory pore are present on the anterior part of the body. On the other hand, potential species-specific features regarding the latter larval stage are discussed. Finally, some G. turgidum life cycle considerations are portrayed.     

Evaluation of mosquitocidal activity of essential oil and sesquiterpenes from leaves of <Emphasis Type="Italic">Chloroxylon swietenia</Emphasis> DC.

Growing awareness in using ecofriendly and biologically compatible phytoconstituents as natural insecticides and repellents for the safety of life and ecological balance led to conscientious efforts by scientists all over the world to search for alternative sources of plant derivatives for effective use as mosquitocides. Encouraged by this, the essential oil and the sesquiterpenes isolated from the leaves of Chloroxylon swietenia DC. were screened for mosquitocidal activity by fumigant toxicity against three mosquito species, Anopheles gambiae, Culex quinquefasciatus and Aedes aegypti. The essential oil had pronounced mosquitocidal activity with LD₅₀ of 1.0, 1.2 and 1.7 × 10⁻³ mg/cm⁻³, respectively, for the three vector species. Furthermore, the major sesquiterpenes were tested at different doses, which again showed varying levels of toxicity. However, germacrene D performed better and proved to be the potential candidate with LD₅₀ values of 1.8–2.8 × 10⁻³ mg/cm⁻³ followed by pregeijerene and geijerene. Nevertheless, the oil and the isolated compounds were particularly active against A. gambiae. The essential oil from the leaves was obtained by hydrodistillation, and the chemical composition was determined by GC and GC–MS. The main compounds identified were limonene, germacrene D, geijerene, pregeijerene, trans-β-ocimene and methyl eugenol. The present study indicates that the oil and the isolated compounds of C. swietenia displayed remarkable mosquitocidal activity suggesting that the method could be extended for future field trials in various mosquito control programmes, and the results are compared with synthetic insecticides.     

L3L4ES antigen and secretagogues induce histamine release from porcine peripheral blood basophils after <Emphasis Type="Italic">Ascaris suum</Emphasis> infection

The aim of this paper was to investigate the role of porcine basophils in protective immunity. Experimental pigs were infected with 10³ Ascaris suum eggs daily for 21 days. Control pigs were maintained helminth-free. Circulating porcine basophils were isolated from the anticoagulated whole blood of A. suum-infected and noninfected pigs by dextran (4.5%) sedimentation of erythrocytes or by the centrifugation of dextran-isolated leukocytes through discontinuous Percoll gradients. Results showed that 2.2% of the isolated leukocytes, stained with May-Grunwald Giemsa, were basophils. Each basophil from infected pigs contained 1.30 × 10⁻² to 1.20 × 10⁻¹ pg of histamine. Peripheral blood basophils (PBBs) from infected swine released 49% specific histamine when induced with A. suum-derived antigen (L3L4ES), 55% with anti-immunoglobulin G, and 62% with calcium ionophore A23l87. During A. suum infection, the number of isolated basophils and histamine levels peaked at 14 to 21 days postinfection and then showed a significant decrease. Percent-specific histamine released from PBBs by infected swine was significantly greater than that released by control pigs. The L3L4ES antigen and secretagogues effectively induced specific/nonspecific histamine release from PBBs and should facilitate future investigations of porcine basophils.     

Growth rate and trapping efficacy of nematode-trapping fungi under constant and fluctuating temperatures

The effect of temperature on radial growth and predatory activity of different isolates of nematode-trapping fungi was assessed. Four isolates of Duddingtonia flagrans and one isolate of Arthrobotrys oligospora were inoculated on petri dishes containing either corn-meal agar (CMA) or faecal agar and then incubated for 14 days under three different constant and fluctuating temperature regimes. The radial growth was similar on the two substrates at each temperature regime. All fungal isolates showed a higher growth rate at a constant 20 °C. At 10° and 15 °C, all D. flagrans isolates showed very similar patterns of radial growth at both constant and fluctuating temperatures. At 20 °C, they grew significantly faster at constant than at fluctuating temperatures. A. oligospora grew significantly faster than all D. flagrans isolates except when incubated at a fluctuating 20 °C. Spores of each fungal isolate were added to faecal cultures containing eggs of Cooperia oncophora at a concentration of 6250 spores/g faeces. The cultures were incubated for 14 days at the same temperature regimes described above. Control faeces (without fungal material) were also cultured. More larvae were recovered from the fungus-treated cultures incubated at a constant 10° or 15 °C than from those incubated at the respective fluctuating temperatures, except for one D. flagrans isolate. Incubation at 20 °C showed the opposite effect. The general reduction observed in the number of nematode larvae due to fungal trapping was 18–25% and 48–80% for a constant and fluctuating 10 °C, 70–96% and 93–95% for a constant and fluctuating 15 °C, and 63–98% and 0–25% for a constant and fluctuating 20 °C, respectively. Received: 15 December 1998 / Accepted: 16 February 1999     

Resistance of different fungal structures of Duddingtonia flagrans to the digestive process and predatory ability on larvae of Haemonchus contortus and Strongyloides papillosus in goat feces

The dynamics of the passage of conidia, chlamydospores, and mycelia of the fungus Duddingtonia flagrans through the digestive tracts of goats was evaluated. Four groups with five goats each were formed. In the group conidia, each animal received 1 × 10⁶ D. flagrans conidia per kilogram of live weight. In the group chlamydospore, each animal received 1 × 10⁶ chlamydospores per kilogram of live weight. In the group mycelia, each animal received 1 g of mycelium mass per kilogram of live weight. In the control group, the animals received no fungal structure. Feces were obtained 3 h before and 12, 24, 30, 36, 42, 48, 60, 72, 84, and 96 h after the inoculation. The feces were placed in Petri dishes containing water-agar. The Petri dishes were examined to detect the fungus and trapped nematodes. A second trial evaluated the effect of the fungal structures on the number of gastrointestinal larvae of Haemonchus contortus and Strongyloides papillosus harvested from the fecal cultures of the goats. The feces were obtained from the goats in the 12–24, 24–30, 30–36, 42–48, 60–72, 72–84, and 84–96 intervals after the inoculation. D. flagrans survived the digestive process of the goats and maintained its predatory activity, being observed from 12 to 96 h before inoculation in the animals that received chlamydospores and conidia.     

Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant ( Elephas maximus ) and Man ( Homo sapiens )

Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, P _d of water in RBC was measured by using a Mn²⁺-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of P _d were, in the case of elephant RBC, 3.2 × 10⁻³ cm/s at 25 °C, 3.9 × 10⁻³ cm/s at 30 °C, 5.2 × 10⁻³ cm/s at 37 °C and 6.5 × 10⁻³ cm/s at 42 °C; all values were significantly lower than the corresponding values of P _d for human RBC, namely 4.3 × 10⁻³ cm/s at 25 °C, 5.2 × 10⁻³ cm/s at 30 °C, 6.1 × 10⁻³ cm/s at 37 °C, 7.8 × 10⁻³ cm/s at 42°C. The maximal inhibition of P _d (56%) was reached in 30 min at 37 °C with 2 mm p-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10⁻³ cm/s for elephant and 2.6 × 10⁻³ cm/s for human RBC. The values of the activation energy for water permeability (E _a,d ) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.