Effect of oregano (<Emphasis Type="Italic">Origanum vulgare</Emphasis> L.) and thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> L.) essential oils on <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> (Protozoa: Kinetoplastida) growth and ultrastructure

In the present work, we have investigated the effect of essential oils obtained from Origanum vulgare L. (oregano) and Thymus vulgaris L. (thyme) on growth and ultrastructure of diverse evolutive forms of Trypanosoma cruzi. Culture epimastigotes and bloodstream trypomastigotes were incubated for 24 h with different concentrations of oregano or thyme essential oils and with thymol (the main constituent of thyme), and the inhibitory concentration (IC)₅₀ was determined by cell counting. Crude extract of oregano essential oil inhibited epimastigote growth (IC₅₀/24 h = 175 μg/ml) and also induced trypomastigote lysis (IC₅₀/24 h = 115 μg/ml). Thyme essential oil presented IC₅₀/24 h values of 77 μg/ml for epimastigotes and 38 μg/ml for trypomastigotes, while treatment with thymol resulted in an IC₅₀/24 h of 62 μg/ml for epimastigotes and 53 μg/ml for trypomastigotes. Scanning electron microscopy of treated cells showed few morphological alterations at the plasma membrane. Observation by transmission electron microscopy showed cytoplasmic swelling with occasional morphological alterations in plasma and flagellar membrane. Our data indicate that oregano and thyme essential oils are effective against T. cruzi, with higher activity of thyme, and that thymol may be the main component responsible for the trypanocidal activity.     

Oviposition-altering and ovicidal potentials of five essential oils against female adults of the dengue vector, <Emphasis Type="BoldItalic">Aedes aegypti</Emphasis> L.

The oviposition deterrence and ovicidal potential of five different essential oils, peppermint oil (Mentha piperita), basil oil (Ocimum basilicum), rosemary oil (Rosemarinus officinalis), citronella oil (Cymbopogon nardus), and celery seed oil (Apium graveolens), were assessed against female adults of the dengue vector, Aedes aegypti L. Multiple concentration tests were carried out where cups containing 1 mL of different concentrations (100%, 10%, 1%, 0.1%) of the oils and 199 mL of water were used for oviposition. The number of eggs laid and the larvae hatched in each cup were scored to evaluate the oviposition deterrent and ovicidal potentials of the oils. Our investigations revealed that the addition of 100% oil (pure oil) caused complete oviposition deterrence except in A. graveolens which resulted in 75% effective repellency. The use of 10% oil resulted in the maximum deterrence of 97.5% as shown by the M. piperita oil while other oils caused 36–97% oviposition deterrence as against the control. The oviposition medium with 1% oil showed decreased deterrent potential with 30–64% effective repellency, the M. piperita oil being exceptional. However, as the concentrations of the oil were reduced further to 0.1%, the least effective oil observed was A. graveolens (25% ER). Also, the M. piperita oil showed much reduced activity (40%) as compared to the control, while the other oils exhibited 51–58% repellency to oviposition. The studies on the ovicidal effects of these oils revealed that the eggs laid in the water with 100% essential oils did not hatch at all, whereas when 10% oils were used, only the R. officinalis oil resulted in 28% egg hatch. At lower concentrations (1%), the oils of M. piperita, O. basilicum, and C. nardus showed complete egg mortality while those of A. graveolens and R. officinalis resulted in 71% and 34% egg hatches, respectively. When used at 0.1%, the O. basilicum oil was found to be the only effective oil with 100% egg mortality, whereas other oils resulted in 16–76% egg mortality, the least mortality caused by the A. graveolens oil. These results suggest that these essential oils can be employed in a resistance-management program against A. aegypti. Further detailed research is needed to identify the active ingredient in the extracts and implement the effective mosquito management program.     

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.     

Visualization of chromosomes in the binucleate intestinal parasite Giardia lamblia

Mitosis of Giardia lamblia is a complex and rapid event that is poorly understood at the cellular and molecular levels. Therefore, we conducted this study to determine (1) whether the two nuclei have similar or different chromosomes, (2) the number of chromosomes of G. lamblia, and (3) the morphology and karyotype of the chromosomes. Trophozoites of the C2 and WB strains of G. lamblia were grown in modified TYI-S-33 medium at 37°C. The trophozoites were collected, and sample slides were prepared for conventional light and scanning electron microscopy. Light microscopy revealed five pairs of chromosomes. The chromosomes were approximately 0.64–0.94 μm long with a short rod-like shape and were usually arranged in pairs. Scanning electron microscopy yielded similar findings, and 10 chromosomes could be seen in each nucleus. Thus, the chromosome number of G. lamblia is 2n = 10. Chromosomes in pair 1 are submetacentric chromosomes, while pairs 2–5 are telocentric chromosomes. The present study shows that G. lamblia trophozoites have typical condensed chromosomes during mitosis and contains five pairs of chromosomes. The karyogram shows good fit to the formula 2n = 10 = 2sm + 8t revealed by scanning electron microscopy.     

Chemical composition and larvicidal evaluation of Mentha, Salvia, and Melissa essential oils against the West Nile virus mosquito Culex pipiens

The volatile metabolites of wild-growing Mentha spicata, M. longifolia, M. suaveolens, Melissa officinalis, Salvia fruticosa, S. pomifera subsp. calycina, and S. pomifera subsp. pomifera from Greece were determined by gas chromatography and gas chromatography–mass spectrometry. The insecticidal properties of the analyzed essential oils were screened on Culex pipiens larvae. Additionally two of the main components of the essential oils, piperitenone oxide and 1,8-cineole were assayed against C. pipiens in order to define the affiliation between them and the larvicidal properties of the oils. The most effective oils were M. suaveolens (major constituent piperitenone oxide, 62.4%), M. spicata (piperitenone oxide, 35.7% and 1,8-cineole, 14.5%) and M. longifolia—Central Greece (piperitenone oxide, 33.4%; 1,8-cineole, 24.5% and trans-piperitone epoxide, 17.4%), which exhibited LC₅₀ values ranging from 47.88 to 59.33 mg l⁻¹. Medium activity revealed the oils of M. officinalis (terpin-4-ol, 15.8%; caryophyllene oxide, 13.2%; sabinene, 12.9%; β-pinene, 12.1%; and trans-caryophyllene, 10.2%), M. longifolia—Southern Greece (carvone, 54.7% and limonene 20.0%), S. pomifera subsp. pomifera (trans-caryophyllene, 22.5% and trans-thujone, 21.0%), S. pomifera subsp. calycina—West Southern Greece (trans-thujone, 56.1% and 1,8-cineole, 10.4%), and S. fruticosa—population 2 (camphor, 23.1%; α-pinene, 12.7%; and borneol, 12.6%), with LC₅₀ values ranging from 78.28 to 91.45 mg l⁻¹. S. pomifera subsp. calycina (Central Greece) essential oil (trans-thujone, 26.5% and cis-thujone, 12.0%) presented rather low activity (LC₅₀ values 140.42 mg l⁻¹), while S. fruticosa—population 1 (1,8-cineole, 31.4% and camphor, 22.6%) was the only inactive oil. Additionally, the constituent piperitenone oxide was found to be highly active (LC₅₀ values 9.95 mg l⁻¹), whereas 1,8-cineole revealed no toxicity.     

Characterization of Giardia lamblia groups A and B from North India by isoenzyme and random amplified polymorphic DNA analysis

Giardia lamblia ( syn. G. intestinalis) infection in young adults leads to acute/chronic diarrhea in some individuals and is asymptomatic in others. Recently, G. lamblia strains have been characterized as group A (symptomatic) and group B (asymptomatic or control) by advanced isoenzyme and molecular biology studies. In the present brief pilot study, ten G. lamblia isolates obtained from five symptomatic (group A) and five asymptomatic (group B) persons were characterized by isoenzyme and random amplified polymorphic DNA (RAPD) analysis. Isoenzyme analysis demonstrated remarkable homogeneity in seven enzyme patterns, the exception, being that of phosphoglucomutase, for which two zymodemes (I and III) were observed. In contrast, RAPD analysis showed homogeneity for eight primers; exceptions were two primers, A₀₂ and B₀₅, which separated group A G. lamblia isolates into two rapdemes (A_R1 and A_R2) and group B G. lamblia isolates into four rapdemes (B_R1, B_R2, B_R3 and B_R4). Further phenetic analysis showed average genetic distances of 0.105 within group A and 0.121 within group B G. lamblia isolates according to Jaccord's distance scale, which suggests that both lineages appear to consist of a range of variants with no significant (P < 0.05) genetic diversity. The two techniques demonstrated a positive association with regard to differentiation between group A and group B G. lamblia isolates. These very preliminary results indicate that RAPD analysis could be a potentially useful substitute for isoenzyme analysis. Received: 4 December 1998 / Accepted: 21 December 1998     

Eucalyptus essential oil toxicity against permethrin-resistant Pediculus humanus capitis (Phthiraptera: Pediculidae)

During the past decades, chemical control against the head louse Pediculus humanus capitis De Geer has been based in the application of products containing permethrin. The repetitive overuse of pediculicides has resulted in the development of high levels of resistance to one or more of these products worldwide. Essential oils obtained from aromatic plants like Eucalyptus are good and safe alternatives due to their low toxicity to mammals and easy biodegradability. In the present study, we reported the chemical composition of Eucalyptus dunnii and Eucalyptus gunni, and the fumigant activity of five Eucalyptus essential oils and their main compounds against permethrin-resistant head lice from Argentina. The most effective essential oils were Eucalyptus sideroxylon, Eucalyptus globulus ssp globulus, and Eucalyptus globulus ssp maidenii, with knockdown time 50% (KT₅₀) values of 24.75, 27.73, and 31.39 min. A linear regression analysis between percentage of 1,8-Cineole and KT₅₀ values of the essential oils showed a significant correlation at a p < 0.01. Since Eucalyptus essential oils showed to be effective against head lice and are classified as safer compounds, they can be employed into pediculicide formulations.     

Acaricidal efficacy of thymol on engorged nymphs and females of Rhipicephalus sanguineus (Latreille, 1808) (Acari: Ixodidae)

The present work was aimed at evaluating the “in vitro” efficacy of different concentrations of thymol on engorged nymphs and females of Rhipicephalus sanguineus. The nymphs were separated in seven groups and immersed in different concentrations of thymol (0.25%, 0.5%, 1.0%, 1.5%, and 2.0%) for 5 min. A control group was established (water + dimethylsulfoxide) together with a positive control group (Amitraz*), and mortality was evaluated after 15 days. In order to perform the experiment with females, the same methodology was used, and the following concentrations were tested: 1.0%, 1.5% and 2.0%. After 5 min of immersion, the engorged females were fixed on Petri dishes with the aid of a double-sided adhesive tape and kept in a climatized room regulated at 27 ± 1°C and UR >80 ± 10%, and different parameters referring to the biology of the non-parasitary phase were evaluated daily. The values found for thymol efficacy on nymphs were 0.0%, 100%, 100%, 100%, and 100% in concentrations of 0.25%, 0.5%, 1.0%, 1.5%, and 2.0%, respectively. In the experiment with engorged females, thymol did not induce any significant alterations (p < 0.05) in the parameters of weight alteration, egg mass weight, pre-oviposition period, hatching percentage, egg production index and nutritional index; however, it affected the engorged females final weight in all treatments (p > 0.05). The concentration of thymol 2% was the one that showed a better efficacy (41%). It was concluded that thymol had a more accentuated deleterious effect on engorged nymphs, and it might be a promising supporting agent for the control of this ixodid.     

Principal intestinal parasites of dogs in Tirana,Albania

From 2004 to 2009, the digestive tracts of 111 dogs from suburban areas around Tirana, Albania, were examined for intestinal helminths. In addition, rectal faecal samples of all dogs were examined for protozoan infections and 48 faecal samples from dogs >6 months of age were processed with the Baermann technique to test for the excretion of lungworm larvae. The heart and pulmonary arteries of 30 dogs >6 months of age also were examined for nematode parasites. The intestinal parasite fauna of the dogs included three protozoan species (Cystoisospora canis, Cystoisospora ohioensis/burrowsi, Sarcocystis spp.), three cestode species (Dipylidium caninum, Taenia hydatigena, Echinococcus granulosus), five nematode species (Ancylostoma caninum, Uncinaria stenocephala, Toxocara canis, Toxascaris leonina, Trichuris vulpis) and one acanthocephalan (Centrorhynchus buteonis). Rates of infection were: 15.3% for C. canis, 31.5% for C. ohioensis/burrowsi, 1.8% for Sarcocystis spp., 65.8% for D. caninum, 16.2% for T. hydatigena, 2.7% for E. granulosus (genotype G1), 13.5% for A. caninum, 64.9% for U. stenocephala, 75.7% for T. canis, 0.9% for T. leonina, 21.6% for T. vulpis and 0.9% for C. buteonis. Up to six species of gastrointestinal parasites were found per dog. The 63 ≤6-month-old dogs harboured significantly (p < 0.001) fewer gastrointestinal parasite species concurrently (mean 2.65 ± 1.25 species per animal) than the 48 older animals (mean 3.77 ± 1.45 species per animal). Dogs >6 months of age harboured significantly (p < 0.05) more D. caninum, T. hydatigena, A. caninum, U. stenocephala and T. vulpis compared to younger dogs. Conversely, the younger dogs harboured significantly (p < 0.001) more T. canis than the older ones. There was no difference in the male and female dogs’ counts of individual intestinal helminth species apart from T. hydatigena in dogs >6 months of age: Male dogs harboured significantly (p < 0.05) more tapeworms than female dogs. Based on faecal examination, there was no indication for lungworm infection; however, two adult heartworms (Dirofilaria immitis) were found in the right ventricle of one dog.     

Infectivity, predilection sites, and freeze tolerance of Trichinella spp. in experimentally infected sheep

A total of 36 sheep in groups of 4 were inoculated with 9 isolates of Trichinella and euthanized after 10 weeks. Thereafter, numbers of muscle larvae were determined in 13 different muscles/muscle groups. Muscle larvae were found in high numbers in all four sheep inoculated with T. spiralis, in lower numbers in two sheep inoculated with T. pseudospiralis (USA isolate), and in very low numbers in one sheep inoculated with T. pseudospiralis (USSR isolate) and one inoculated with T. britovi. In infections of high and moderate larval intensity, predilection sites of T. spiralis were the masseter muscles, the tongue, and the diaphragm and those of T. pseudospiralis were the masseter muscle and the neck. In low-intensity infections, muscle larvae were detected only in the diaphragm or in pooled muscle samples. For evaluation of the freeze tolerance of the different Trichinella species in sheep-muscle tissue, samples taken from the filet were stored at +5°, −5°, and −18 °C, respectively. After exposure for 1 and 4 weeks the tissue was digested and the released larvae were inoculated into mice for determination of the reproductive capacity index (RCI). Larvae of both T. spiralis and T. pseudospiralis survived freezing at −5° and −18 °C for 4 weeks. Received: 10 September 1999 / Accepted: 22 October 1999     

Coinfection is an important factor in epidemiological studies: the first serosurvey of the aoudad (<Emphasis Type="Italic">Ammotragus lervia</Emphasis>)

Despite being considered an invasive ungulate outside its native range (North Africa), little information exists regarding the role of the aoudad (also called Barbary sheep, Ammotragus lervia) as a pathogen reservoir. Furthermore, in most epidemiological surveys the potential role of coinfections (e.g. a first infection may make the host more immuno-competent or susceptible against a second pathogen) as a risk factor is often neglected. In this study we first performed a serological survey for selected pathogens (Mycobacterium bovis, M. avium subsp. paratuberculosis, Chlamydophila abortus, bovine viral diarrhoea/border disease viruses (BVDV-BDV), Salmonella spp., Brucella melitensis and Toxoplasma gondii) on free (n = 66) and captive (n = 25) aoudad from south-east Spain. Then, by using Akaike’s information criterion, we evaluated the importance of coinfection in two statistical models that included the effects of population, age, and sex. Our results show that neither free nor captive aoudad had antibodies against Brucella melitensis, Chlamydophila abortus, or BVDV-BDV. However, compared to other wild ungulates in Spain, aoudads have high prevalence of antibodies against M. bovis (free = 49.5%; captive = 8%), very high prevalence of antibodies against M. avium subsp. paratuberculosis (free = 19.4%; captive = 56%), and intermediate prevalence of antibodies against Salmonella spp. (free = 13.4%; captive = 0%) or T. gondii (free = 1.5%; captive = 24%). Although the additive effects of population and age were included in our set of selected models, coinfection was the most influential factor to detect antibodies response against mycobacterials and salmonella infections. The direction of this influence could be exclusion of disease between tuberculosis and paratuberculosis seroreactor animals, or enhanced susceptibility to disease between tuberculosis and salmonella seroreactor animals. In conclusion, we believe that wildlife managers must pay more attention to the potential risk posed by aoudads as hosts (and probably reservoirs) of paratuberculosis and tuberculosis mycobacterials, while epidemiologists should be more aware of coinfection as an important factor in epidemiological surveys, especially in wildlife populations where multiple infections are common.     

Giardiasis in kindergartens: prevalence study in Berlin,Germany, 2006

In Germany, an increase of notified giardiasis was observed between 2002 (n = 3,101) and 2007 (n = 3,651) with 62% of cases acquired in Germany. The highest incidence was reported in 1- to 5-year-olds (2001–2007, 11.5/100,000 on average) and Berlin is one of the most affected states (17.5/100,000). We performed a cross-sectional study in five Berlin kindergartens differing in socioeconomic status and migrant proportion in order to estimate the prevalence and investigate routes of transmission among children under 6 years of age. Stool samples were screened for Giardia lamblia and Cryptosporidium parvum using microscopical and antigen detection. Giardia-positive samples underwent PCR and subtyping. Two hundred two children participated (mean age 3.4 years). We found three girls (1.5%) who tested positive for G. lamblia genotype-A3, all clustering in one kindergarten (prevalence 5.5%). No common source was identified. Two children were symptomatic. Possible dog-to-child transmission was established for one of the symptomatic cases. All contact-tracing results were negative. Other microscopically detected parasites (Blastocystis hominis, Endolimax nana cysts, Entamoeba coli cysts and Iodamoeba bütschlii cysts) were found in 4% of children. In summary, a substantial level of Giardiasis may be prevalent in some of Berlin’s kindergartens, despite standard hygienic measures being followed. The relatively high prevalence in one kindergarten indicates the need for further studies to identify risk factors for children, which may help to guide possible interventions and strategies. Giardiasis should be considered as a differential diagnosis in children with unclear gastrointestinal symptoms. Additional education and training on proper toilet and food hygiene may further reduce the possibility of child-to-child transmission.     

Acaricidal activity of the essential oils from Eucalyptus citriodora and Cymbopogon nardus on larvae of Amblyomma cajennense (Acari: Ixodidae) and Anocentor nitens (Acari: Ixodidae)

The present study evaluated the acaricidal activity of essential oils from Eucalyptus citriodora and Cymbopogon nardus on non-engorged larvae of Amblyomma cajennense and Anocentor nitens. In order to carry out the study, six groups were formed, each concentration being a treatment (6.25%, 12.5%, 25%, and 50%, respectively) and also with the creation of a control group (distilled water) and a positive control (Deltametrine). For each treatment, approximately 100 larvae of these ticks were placed onto filter papers (2 × 2 cm) impregnated with the concentrations used to test. Next, the envelopes were closed bearing inside the filter paper with measurements of 6 × 6 cm. For each group, six repetitions were performed, and after 24 h live and dead larvae were counted. This procedure was carried out for two essential oils on the two species of ticks. For A. cajennense, the acaricide efficacy of E. citriodora oil was of 10.8%, 35.3%, 34.5%, and 53.1%, whereas the efficacy of C. nardus was of 0.0%, 0.0%, 0.0%, and 61.1% at concentrations of 6.25%, 12.5%, 25.0%, and 50.0%, respectively. In relation to A. nitens, the acaricide efficacy of E. citriodora oil was of 20.1%, 84.5%, 89.2%, and 100.0%, whereas the efficacy of C. nardus was of 0.0%, 90.8%, 100.0%, and 100.0% at concentrations of 6.25%, 12.5%, 25.0%, and 50.0%, respectively. The results indicate that the essential oils tested showed a promising acaricidal activity mainly on A. nitens larvae.     

The effects of dihydroartemisinin on Giardia lamblia morphology and cell cycle in vitro

Several anti-Giardia drugs, such as metronidazole, tinidazole, mebendazole, albendazole and furazolidone, are usually effective but have severe side effects and potential toxicity. An urgent need exists for more effective and less toxic agents that can act against this protozoan. For this purpose, the effects of dihydroartemisinin (DHA) on Giardia lamblia were investigated in vitro. Axenically grown G. lamblia trophozoites were treated with DHA (LD₅₀ = 200 μg/mL) at different time intervals. The morphological and ultrastructural changes of the treated trophozoites were observed by both light and transmission electron microscopy (TEM). Changes in the cell cycle of the treated cells were observed by flow cytometry. By light microscopy, we observed that DHA-treated trophozoites were detached from the wall of the culture tube and shown bradypraxia and bubbles in the dorsal and ventral surfaces. Ultrastructural observations by TEM revealed that DHA promoted modifications of the cell shape, pronounced dorsal vesiculation, plasma membrane blebbing, disaggregation of ribosomes, depletion of cytoplasmic matrix and heavy deposition of electron-dense precipitates on the cytoplasm and nucleus. The main changes observed in the treated group included the following: (1) trophozoites were rounder in shape and the endoplasmic reticulum was dilated, (2) enlarged trophozoites contained lamella structures and deformed nuclei, (3) trophozoites displayed dissolved cytoplasm with large vacuole spaces or decreased cytoplasmic volume, (4) adhesive disc bubbles or the lamella structures of cytoplasm were clearly observed, and (5) cell division was arrested. Using microscopy and cytometry techniques, we demonstrate that changes in G. lamblia morphology and cell cycle state were induced by DHA     

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.     

Possible role of calmodulin in excystation of Giardia lamblia

The protozoan Giardia lamblia initiates infection when trophozoites emerge from a cyst in the hosts by the excystation process. Although this process is crucial to the initiation of infection by G. lamblia, little is known about its regulation. To study the possible involvement of calmodulin (CaM) in excystation we tested the effect of several CaM antagonists (TFP, W-7, and W-5) on this cellular function. Except for W-5 the rest of these compounds inhibited excystation. The protein kinase C inhibitor H-7 had no effect on excystation, suggesting that CaM antagonists acted by selectively inhibiting CaM. Furthermore, CaM was redistributed after the induction of excystation and there was an increase in its fluorescence and activity. These results suggest that a CaM-dependent process is involved in G. lamblia excystation. Received: 10 March 1998 / Accepted: 22 April 1998     

Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Pará State, Amazon, Brazil

A cross-sectional study was conducted to determine the occurrence of anti-Toxoplasma gondii, anti-Neospora caninum, and anti- Leishmania chagasi antibodies in dogs of the state of Pará, Brazil. For this purpose, 129 blood samples were collected from dogs of different ages and gender. Samples of 72 dogs were collected from 39 rural properties from 19 municipalities, and 57 samples were from stray dogs, collected after captivity by the Center of Zoonosis Control from the municipality of Santarém. The sera were analyzed for anti-T. gondii and anti-N. caninum antibodies by indirect fluorescent antibody tests with cutoff values of 1:16 and 1:50, respectively. For the presence of L. chagasi antibodies, enzyme-linked immunosorbent assay was used and positive results were confirmed by immunochromatographic method using the recombinant antigen K39. Of the total of 129 dogs, 90 (69.8%) were positive for T. gondii, 16 (12.4%) for N. caninum, and 30 (23.3%) for L. chagasi. Antibodies for all three parasites were found simultaneously in seven dogs (5.4%), mostly in urban dogs (six of seven). No association was observed related to gender and location (urban or rural) of dogs and occurrence of N. caninum and T. gondii antibodies although, regarding L. chagasi, higher prevalence was found in females (P < 0.02) and in dogs from urban location (P < 0.001). From the 39 farms, in 30 (76.9%) at least one dog was positive for T. gondii or N. caninum or both. Higher occurrence of Leishmania antibodies was observed in N. caninum-negative dogs (P < 0.05).     

Cellular effects induced by Inula graveolens and Santolina corsica essential oils on Staphylococcus aureus

This study was conducted to evaluate the antibacterial activity of Inula graveolens and Santolina corsica essential oils on Staphylococcus aureus and investigate their effects at the cellular level. The mode of inhibition of both essential oils against S. aureus ATCC 6538P (CIP 53.156) was assessed by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The effects of time and treatment dose on cell viability were determined by time-kill and bacteriolysis assays. Marked structural changes were observed by transmission electron microscopy (TEM). A bactericidal mode of inhibition was established for both essential oils, which rapidly reduced the cell viability of S. aureus at their MIC (5 mg·ml^-1). No lysis occurred after treatments with the MIC and eight times the MIC of each essential oil. Invaginations of the plasmic membrane with thickenings of the cell wall as well as an aggregation of the cytoplasmic contents were observed in S. aureus cells treated with the MIC of both essential oils. These results suggest that the cytoplasmic membrane and the cell wall are involved in the toxic action of Inula graveolens and Santolina corsica essential oils.     

Antigiardial activity of <Emphasis Type="Italic">Ocimum basilicum</Emphasis> essential oil

In this study, we investigated the effects of Ocimum basilicum essential oil on Giardia lamblia and on the modulation of the interaction of these parasites by peritoneal mouse macrophage. The essential oil (2 mg/ml) and its purified substances demonstrated antigiardial activity. Linalool (300 μg/ml), however, was able to kill 100% parasites after 1 h of incubation, which demonstrates its high antigiardial potential. Pretreatment of peritoneal mouse macrophages with 2 mg/ml essential oil dilution reduced in 79% the association index between these macrophages and G. lamblia, with a concomitant increase by 153% on nitric oxide production by the G. lamblia-ingested macrophages. The protein profiles and proteolitic activity of these parasite trophozoites, previously treated or not with 2 mg/ml essential oil or with the purified fractions, were also determined. After 1 and 2 h of incubation, proteins of lysates and culture supernatants revealed significant differences in bands patterns when compared to controls. Besides, the proteolitic activity, mainly of cysteine proteases, was clearly inhibited by the essential oil (2 mg/ml) and the purified linalool (300 μg/ml). These results suggest that, with G. lamblia, the essential oil from O. basilicum and its purified compounds, specially linalool, have a potent antimicrobial activity. Igor de Almeida and Daniela Sales Alviano contributed equally to this study.