Kudoa sciaenae (Myxozoa: Multivalvulidae) cysts distribution in the somatic muscles of Stellifer minor (Tschudi, 1844) (Pisces: Sciaenidae)

The distribution of Kudoa sciaenae cysts (Myxozoa), in terms of intensity and prevalence, in the somatic muscles of the sciaenid Stellifer minor, shows an apparent preference for the anterior body region, including the head. The observed preference seems to be a consequence of the differential distribution of muscle mass, in the defined area, because when density (cyst/g dry muscle), is considered, all the somatic areas, but not cephalic area, do no show significant differences in terms of mean intensity and prevalence.

On the current existence of a coccidial line of development in the malaria parasites: a theory

Most opinion favors the origin of the malaria parasites from a coccidial ancestor. It is assumed that whatever the process through which the coccidia differentiated into a Plasmodium this phenomenon very probably occured millions of year ago, and during that differentiation process the original coccidia vanished. Therefore it has never repeated. At the light of some experiments the existence, at the present time, of a coccidial cycle of development in the malaria parasites, is proposed. The conection routes and mechanisms through which the malaria parasite changes to a coccidial life, and the routes in reverse are exposed. Transmission of the malaria-coccidial forms is suggested.

The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages

We characterized the Plasmodium falciparum antigen 332 (Ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. The corresponding Pf332 gene has been located in the subtelomeric region of chromosome 11. Furthermore, it is present in all strais so far analyzed and shows marked restriction length fragment polymorphism. Partial sequence and restriction endonuclease digestion of cloned fragments revealed that the Pf332 gene is composed of highly degenerated repeats rich is glutamic acid. Mung been nuclease digestion and Northern blot analysis suggested that Pf332 gene codes for a protein of about 700 kDa. These data were further confirmed by Western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the Ag332. Confocal immunofluorescence showed that Ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. In addition, Ag332 was detected on the surface of monkey erythrocytes infected with Plasmodium falciparum.

Mechanisms of protective immunity against asexual blood stages of Plasmodium falciparum in the experimental host Saimiri

In the Saimiri monkey, an experimental host for human malaria, acquired protection against Plasmodium falciparum blood stages depends on the IgG antibody populations developed. In vivo protective anti-falciparum activity of IgG antibodies is correlated with the in vivo opsonizing activity promoting phagocytosis of parasited red bloood cells. In contrast, non protective antibodies inhibit this mechanism by competing at the target level. A similar phenomenon can be and human infection. Anti-cytoadherent and anti-rosette antibodies developed by Saimiri and humans prevent the development of physiopathological events like cerebral malaria which can also occur in this experimental host. Furthermore, transfer to protective human anti-falciparum IgG antibodies into infected Saimiri monkeys exerts an anti parasite activity as efficient as that observed when it is transfered into acute falciparum malaria patients, making the Saimiri an even more attractive host. Studies on the role of immunocompetent cells in the protective immune reponse are still in their infancy, however the existance of a restricted polymorphism of MHC II class molecules in the Saimiri confers additional theoretical and practical importance to this model.

Mechanisms of immune protection in the asexual blood stage infection by Plasmodium falciparum: analysis by in vitro and ex-vivo assays

Mechanisms of immune protection against the asexual blood stage infection by Plasmodium falciparum are reviewed. Recent studies of two independent lines of research developed at the Institute Pasteur, in humans and primate infections clearly indicate an obligatory interaction of antibodies and effector cells to express the anti-parasitic effect.

Establishment and Characterization of a Cell Line from the Mosquito Anopheles albimanus (Diptera: Culicidae)

A new cell line designated LSB-AA695BB, was established from embryos of the mosquito Anopheles albimanus. The primary culture was initiated in April, 1995, and the first passage was made 48 days later. Serial subcultures of the cells have been carried through 90 passages from Abril 1995 to February 1996. The cells were grown at 28°C in MK/VP12 medium, supplemented with 20% fetal bovine serum; the pH tolerance ranged between 6.8 to 7.0. The cells have also been adapted to MM/VP12 medium under the same pH, temperature and serum concentration. The majority of the cells were a fibroblast-type. Isozyme characterization showed a pattern similar to that of An. albimanus pupae and adults but distinct from Ae. taeniorhynchus and Ae. albopictus (C6/36) mosquito cell lines. The culture was shown to be free of mycoplasma, bacteria and fungi. Microsporidia contamination of transovarial transmission was controlled with 6.0 mg/ml of albendazole

Establishment and characterization of a new continuous cell line from Lutzomyia longipalpis (Diptera: Psychodidae) and its susceptibility to infections with arboviruses and Leishmania chagasi

Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28ºC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.

A new continuous cell line from the mosquito Psorophora confinnis (Diptera: Culicidae) and its susceptibility to infections with some arboviruses

A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20% fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10% fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma.

Laboratory diagnosis of Schistosomiasis in areas of low transmission: a review of a line of research

After 57 years of successful control of schistosomiasis in Venezuela, the prevalence and intensity of infection have declined. Approximately 80% of the individuals eliminate less than 100 eggs/g of stools, therefore morbidity is mild and the majority are asymptomatic. The sensitivity of Kato-Katz decreases to approximately 60%. Available serological methods for the detection of circulating antigens only reach a 70% of sensitivity. Tests based on the detection of antibodies by immunoenzymatic assays have been improved. The circumoval precipitine test has shown a high sensitivity (97%), specificity (100%), and correlation with oviposition, being considered the best confirmatory diagnostic test. Additionally to the classical immunoenzymatic assays, the development of the alkaline phosphatase immunoassay, allowed to reach a 100% specificity with an 89% sensitivity. Recently, we have developed a modified ELISA in which the soluble egg antigen is treated with sodium metaperiodate (SMP-ELISA) in order to eliminate the glycosilated epitopes responsible for the false positive reactions. The specificity and sensitivity reaches 97% and 99%, respectively. Synthetic peptides from the excretory-secretory enzymes, cathepsin B (Sm31) legumain (Sm32) and cathepsin D (Sm45), have been synthesized. The combination of two peptides derived from the Sm31 have been evaluated, reaching a sensitivity of 96% when analyzed independently and with a 100% specificity. Antibodies raised in rabbits against peptides derived from the Sm31 and Sm32 are currently evaluated in two different antigen-capture-based assays. The development of a simple, cheap and reliable test that correlates with parasite activity is a major goal.

Evaluation of indirect susceptibility testing of Mycobacterium tuberculosis to the first- and second-line, and alternative drugs by the newer MB/BacT system

In order to evaluate the Organon Teknika MB/BacT system used for testing indirect susceptibility to the alternative drugs ofloxacin (OFLO), amikacin (AMI), and rifabutin (RIF), and to the usual drugs of standard treatment regimes such as rifampin (RMP), isoniazid (INH), pyrazinamide (PZA), streptomycin (SM), ethambutol (EMB), and ethionamide (ETH), cultures of clinical specimens from 117 patients with pulmonary tuberculosis under multidrug-resistant investigation, admitted sequentially for examination from 2001 to 2002, were studied. Fifty of the Mycobacterium tuberculosis cultures were inoculated into the gold-standard BACTEC 460 TB (Becton Dickinson) for studying resistance to AMI, RIF, and OFLO, and the remaining 67 were inoculated into Lowenstein Jensen (LJ) medium (the gold standard currently used in Brazil) for studying resistance to RMP, INH, PZA, SM, EMB, and ETH. We observed 100% sensitivity for AMI (80.8-100), RIF (80.8-100), and OFLO (78.1-100); and 100% specificity for AMI (85.4-100), RIF (85.4-100), and OFLO (86.7-100) compared to the BACTEC system. Comparing the results obtained in LJ we observed 100% sensitivity for RMP (80-100), followed by INH - 95% (81.8-99.1), EMB - 94.7% (71.9-99.7), and 100% specificity for all drugs tested except for PZA - 98.3 (89.5-99.9) at 95% confidence interval. The results showed a high level of accuracy and demonstrated that the fully automated, non-radiometric MB/BacT system is indicated for routine use in susceptibility testing in public health laboratories.

Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN) and mouse neutralization test (MNT) to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER) cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125) as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5) cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100) and r = 0.9307 for cattle sera (n = 99), as well as good specificity (94.7%), sensitivity (87.5%), and agreement (96.6%) were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.