Leishmania major: Parasite Interactions Suggesting Sexuality

In five experiments, Leishmania (Leishmania) major (MRHO/SU/59/P-strain) grew poorly when seeded in FYTS medium supplemented with 15% fetal calf serum, but presented several peculiar pairs of promastigotes diametrically opposed and attached at their posterior ends (5.8-13.5%). As seen in Giemsa-stained smears, a ring-like line and/or an enlargement, generally occurred at the parasite junction. A close proximity of nuclei, which sometimes were difficult to distinguish from each other, was also observed at this junction. Several of these pairs appeared to be composed of fused cells in which the nuclei could be apparently fused, as shown by fluorescence microscopy to detect ß-tubulin and DNA, and by scanning electron microscopy. Under other culture conditions these pairs were absent or occurred at very low rates (0.2-2.2%). Such pairs differ markedly from longitudinally dividing cells and resemble those described in two other Leishmania species, as well as in Herpetomonas megaseliae and Phytomonas davidi, suggesting steps of a putative sexual process

Scanning Electron Microscopy of Lagochilascaris minor Leiper, 1909 (Nematoda: Ascarididae)

Lagochilascaris minor Leiper, 1909 is a parasitic nematode with its biological cycle still unknown, even though it was found in humans, domestic and silvatic animals. Adult worms, collected by surgical drainage from a human patient from the State of Pará, Brazil, were micrographed using a scanning electron microscope. Morphological aspects of males and females such as cephalic structures, caudal papillae and cuticular patterns were analyzed and compared with the previous descriptions adding new data for the identification of this species.

Evaluation of DNA Recombinant Methodologies for the Diagnosis of Plasmodium falciparum and their Comparison with the Microscopy Assay

Since 1984, DNA tests based on the highly repeated subtelomeric sequences of Plasmodium falciparum (rep 20) have been frequently used in malaria diagnosis. Rep 20 is very specific for this parasite, and is made of 21 bp units, organized in repeated blocks with direct and inverted orientation. Based in this particular organization, we selected a unique consensus oligonucleotide (pf-21) to drive a PCR reaction coupled to hybridization to non-radioactive labeled probes. The pf-21 unique oligo PCR (pf-21-I) assay produced DNA amplification fingerprints when was applied on purified P. falciparum DNA samples (Brazil and Colombia), as well as in patient's blood samples from a large area of Venezuela. The performance of the Pf-21-I assay was compared against Giemsa stained thick blood smears from samples collected at a malaria endemic area of the Bolívar State, Venezuela, at the field station of Malariología in Tumeremo. Coupled to non-radioactive hybridization the pf-21-I performed better than the traditional microscopic method with a r=1.7:1. In the case of mixed infections the r value of P. falciparum detection increased to 2.5:1. The increased diagnostic sensitivity of the test produced with this homologous oligonucleotide could provide an alternative to the epidemiological diagnosis of P. falciparum being currently used in Venezuela endemic areas, where low parasitemia levels and asymptomatic malaria are frequent. In addition, the DNA fingerprint could be tested in molecular population studies

On a New Species of Hysterothylacium (Nematoda: Anisakidae) from Cauque mauleanum (Pisces: Atherinidae) by Brightfield and Scanning Electron Microscopy

Hysterothylacium geschei n. sp. (Nematoda, Anisakidae) is described from the intestine of Cauque mauleanum (Steindachner) (Pisces: Atherinidae) from Lake Panguipulli (39º43'S; 72º13'W), Chile. Eleven (78.6%) out of 14 fish were infected, with a mean intensity (range) of 14.4 (1-55) worms. The new species can be differentiated from the two previously described species of freshwater fishes from South America by the presence of lateral alae, the number of caudal papillae, and the length of the spicules, oesophagus, intestinal caecum, distance vulva-anterior extremity and the length ratio intestinal caecum: ventricular appendix. From the fishes examined in Lake Panguipulli, including the introduced salmonid species Oncorhynchus mykiss (Walbaum) and the authochthonous species Basilichthys australis Eigenmann (Atherinidae) and Percichthys trucha (Valenciennes) (Percichthyidae), only one specimen of P. trucha was found parasitized by a third-stage larva of this species.

Morphological study of adult male worms of Schistosoma mansoni Sambon, 1907 by confocal laser scanning microscopy

Aiming to detail data obtained through brightfield microscopy (BM) on reproductive, excretory and digestive system, specimens of Schistosoma mansoni eight weeks old, were recovered from SW mice, stained with Langeron's carmine and analyzed under a confocal laser scanning microscope CLSM 410 (Carl Zeiss). The reproductive system presented a single and lobate testis, with intercommunications between the lobes without efferent duct. Supernumerary testicular lobe was amorphous and isolated from the normal ones. Collecting tubules (excretory ducts), followed by the excretory bladder, opening to the external media through the excretory pore, were observed at the posterior extremity of the body. In the digestive tract, a cecal swelling was noted at the junction that originates the single cecum. It was concluded that through confocal laser scanning microscopy, new interpretations of morphological structures of S. mansoni worms could be achieved, modifying adopted and current descriptions. The gonad consists of a single lobed testis, similar to that observed in some trematode species. Moreover, the same specimens can be observed either by BM or CLSM, considering that the latter causes only focal and limited damage in tissue structures.

Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV). In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37ºC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.

Posterior spiracles of fourth instar larvae of four species of phlebotomine sand flies (Diptera: Psychodidae) under scanning electron microscopy

In the present study, posterior spiracles of laboratory-reared fourth instar larvae of Lutzomyia longipalpis, L. migonei, L. lenti, and L. whitmani (Diptera: Psychodidae) of the State of Ceará, Brazil, were examined under scanning electron microscopy. The number of papillae of spiracles examined varied according to the species examined, but no intraspecific differences were found. The importance of this structure to sand fly larva identification and phylogeny is commented.

A comparative study on the different staining methods and number of specimens for the detection of acid fast bacilli

The presence of acid fast bacilli in multiple specimens was investigated comparatively with Ziehl-Neelsen (ZN) and fluorescence microscopy (FM) staining in order to determine sensitivity in detecting tuberculosis (TB). A total of 465 specimens obtained from 295 patients were analysed at Harran University Medical School Hospital between March 1998 and March 2000. The culture was employed as the reference method. Sixty-eight patients (23.1%) were diagnosed as having TB by culture. The ZN and FM staining sensitivities were 67.6% (46/68) and 85.2% (58/68) respectively. Two hundred and one patients (68.1%) submitted one specimen to the laboratory. TB positivity was detected in 42 (20.9%) of these patients by culture. The sensitivities of ZN and FM stains were found to be 61% and 83% in these patients. However, in 18 patients (6.1%) who submitted two specimens to the laboratory, the TB was positive in six of them (33.3%) and ZN and FM sensitivities were 66% and 83% respectively. When three specimens or more were collected from the patients (76 patients, 25.8%), TB positivity was determined in 20 of them (26.3%) and the sensitivities were 80% and 92% in the ZN- and FM-stained smears, respectively. Our data indicate that in the diagnosis of TB, FM has greater sensitivity than ZN. In particular, in the case of a single specimen, the diagnostic value of FM is quite significant. It is, therefore, possible to conclude that both ZN and FM staining can be used for the diagnosis of TB when there are more than two specimens. However, if only one or two specimens are available, FM staining is preferable.

Scanning and transmission electron microscopy of the tegument of Paranaella luquei Kohn, Baptista-Farias & Cohen, 2000 (Microcotylidae, Monogenea), parasite of a Brazilian catfish, Hypostomus regani

The surface topography and ultrastructure of the tegument of Paranaella luquei Kohn, Baptista-Farias & Cohen, 2000, a microcotylid monogenean parasite from the gills of Hypostomus regani (Ihering, 1905) (Loricariidae) was studied by scanning (SEM) and transmission electron microscopy (TEM). By SEM, it was observed that the tegument presents transversal ridges, forming folds in the ventral and dorsal surfaces and microvillous-like tegumental projections in the anterior and median regions of body. These projections were also observed by TEM. The tegument is made up of a syncytium delimited by apical and basal plasma membranes, containing inclusion bodies and mitochondria, connected to the nucleated region by means of cytoplasmatic processes. The tegumental cells present a well developed nucleus and cytoplasm containing inclusion bodies, similar to those found on the external layer, mitochondria, rough endoplasmatic reticulum and free ribossomes.

Morphological aspects of Schistosoma mansoni adult worms isolated from nourished and undernourished mice: a comparative analysis by confocal laser scanning microscopy

Malnutrition hampers the course of schistosomiasis mansoni infection just as normal growth of adult worms. A comparative morphometric study on adult specimens (male and female) recovered from undernourished (fed with a low protein diet - regional basic diet) and nourished (rodent commercial laboratory food, NUVILAB) white mice was performed. Tomographic images and morphometric analysis of the oral and ventral suckers, reproductive system and tegument were obtained by means of confocal laser scanning microscopy. Undernourished male specimens presented smaller morphometric values (length and width) of the reproductive system (first, third and last testicular lobes) and thickness of the tegument than controls. Besides that, it was demonstrated that the dorsal surface of the male worms bears large tubercles unevenly distributed, but kept grouped and flat. At the subtegumental region, vacuolated areas were detected. It was concluded that the inadequate nutritional status of the vertebrate host has a negative influence mainly in the reproductive system and topographical somatic development of male adult Schistosoma mansoni, inducing some alterations on the structure of the parasite.

External morphology of sensory structures of fourth instar larvae of neotropical species of phlebotomine sand flies (Diptera: Psychodidae) under scanning electron microscopy

In the present study, some morphological structures of antennae, maxillary palps and caudal setae of fourth instar larvae of laboratory-reared phlebotomine sand flies (Lutzomyia longipalpis, L. migonei, L. evandroi, L. lenti, L. sericea, L. whitmani and L. intermedia) of the State of Ceará, Brazil, were examined under scanning electron microscopy. The antennal structures exhibited considerable variation in the morphology and position. A prominent digitiform distal segment has been observed only on the antenna of species of the subgenus Nyssomyia. The taxonomic relevance of this and other antennal structure is discussed. The papiliform structures found in the maxillae and the porous structures of the caudal setae of all species examined may have chemosensory function. Further studies with transmission electron microscopy are needed to better understand the physiological function of these external structures.

IgM-Immunofluorescence Test as a Diagnostic Tool for Epidemiologic Studies of Schistosomiasis in Low Endemic Areas

The high sensitivity and the ability to diagnose schistosomiasis in a very early phase after infection have indicated the detection of IgM antibodies to Schistosoma mansoni gut antigens by the immunofluorescence test (IgM-IFT) as a useful serological test for epidemiological studies in low endemic areas. When applied in a follow-up study for two years, higher rates of seroconversion from IFT negative to positive were observed during the summer months, suggesting seasonal transmission of schistosomiasis in the rural area of the municipality of Itariri (São Paulo, Brazil). In each survey, blood samples from about 600 schoolchildren were collected on filter paper and submitted to IgM-IFT. When the blood samples were classified for the IgM antibody levels, according to the intensity of fluorescent reaction observed at fluorescence microscopy, and correlated to the egg counts in the Kato-Katz positive patients, no association was observed. This observation might suggest that the intensity of fluorescence observed in the IgM-IFT, as an indicator of IgM antibody levels, could not be an useful seroepidemiological marker for classifying areas of low endemicity according to degrees of infection.

Ultrastructure of Proechinophthirus zumpti (Anoplura, Echinophthiriidae) by scanning electron microscopy

The ultrastructure of Proechinophthirus zumpti Werneck, 1955, mainly the external chorionic features of the egg, is described through electronic microscopy techniques. This species was first cited in Argentina, infesting Arctocephalus australis (Zimmermann, 1873). The morphological adaptations of adults and nymphs are described in both species of Proechinophthirus parasitic on Otariidae: P. fluctus (Ferris, 1916) and P. zumpti.

Comparative study of the stridulatory sulcus, buccula and rostrum of the nymphs of Triatoma guazu Lent & Wygodzinsky, 1979 and Triatoma jurbergi Carcavallo, Galvão & Lent, 1998 by scanning electron microscopy (Hemiptera, Reduviidae)

The ultrastructural morphology of the ventral region of the head (rostrum and buccula) and proesternum (stridulatory sulcus) of nymphs from the 1st to 5th instars of Triatoma guazu Lent & Wygodzinsky, 1979 and Triatoma jurbergi Carcavallo, Galvão & Lent, 1998 was described. Morphological differences between the two species and of the five nymphal stages development of each species were observed. These structures showed systematic differential characteristics of the studied species and may be used to increase their taxonomic range.

Reproductive system abnormalities in Schistosoma mansoni adult worms isolated from Nectomys squamipes (Muridae: Sigmodontinae): brightfield and confocal laser scanning microscopy analysis

Schistosoma mansoni adult worms with genital anomalies isolated from Nectomys squamipes (Muridae: Sigmodontinae) were studied by confocal laser scanning microscopy under the reflected mode. One male without testicular lobes (testicular agenesia/anorchism) and two females, one with an atrophied ovary and another with 17 uterine eggs, were identified. The absence of testicular lobes occurred in a worm presenting otherwise normal male adult characteristics: tegument, tubercles and a gynaecophoric canal with spines. In both female specimens the digestive tube showed a vacuolated appearance, and the specimen with supernumerary uterine eggs exhibited a developing miracidium and an egg with a formed shell. The area of the ventral sucker was similar in both specimens however the tegument thickness, ovary and vitelline glands of the specimen with the atrophied ovary were smaller than those of the one with supernumerary eggs. These reported anomalies in the reproductive system call attention to the need to improve our understanding of genetic regulation and the possible role of environmental influences upon trematode development.