Use of Monoclonal Antibodies for the Identification of Leishmania spp. Isolated from Humans and Wild Rodents in the State of Campeche, Mexico

The genus Leishmania includes 30 described species which infect a wide variety of mammalian hosts. The precise identification of leishmanial parasites at the species level is very important in order to determine whether an organism, causing the disease in a given area, is of the same biotype as that found in suspected mammalian reservoirs. The objectives of the present study were (1) to identify leishmanial parasites isolated from humans and wild rodents from the State of Campeche, an endemic focus of localized cutaneous leishmaniasis (LCL) in southern Mexico, using an indirect immunofluorescent assay (IFA) with monoclonal antibodies (Mabs); and (2) to determine if the parasites of the two types of hosts were of the same biotype. All the wild rodents (six Ototylomys phyllotis, eight Oryzomys melanotis, five Peromyscus yucatanicus and two Sigmodon hispidus) and 96% (24/25) of the human isolates were identified as Leishmania (L.) mexicana confirming that this specific LCL focus is a wild zoonosis. The presence of one human isolate of L. (Viannia) braziliensis in the State of Campeche, confirmed the importance of an accurate taxonomic identification at species level.

In vitro activity of Brazilian strains of the predatory fungi Arthrobotrys spp. on free-living nematodes and infective larvae of Haemonchus placei

In vitro tests were carried out to assess the activity of 26 Brazilian isolates of predatory fungi of the genus Arthrobotrys on a free-living nematode (Panagrellus sp.) and on infective larvae of Haemonchus placei, a parasitic gastrointestinal nematode of cattle. The results showed that the free-living nematode Panagrellus sp. was the most preyed upon, compared to H. placei, for all the fungal treatments. Also, variable predatory capacity was observed for different fungal isolates belonging to the same genus when applied to different nematode species.

Simulium spp. control program in Rio Grande do Sul, Brazil

Insects of the Simuliidae family have been the object of control in Rio Grande do Sul since the 70s. Their constant attacks became a social-economical problem as well as a problem of Public Health, with serious consequences to men and to the economy of the areas in which the insects develop. At first, the control was done with a chemical larvicide Themephos ABATE 500 E, but an imperfect measuring of outflow to determine the quantity of the product made Simulium spp. resistant to it. From 1983 on, following a study of a new method for the outflow measuring, we started to use a biological larvicide Bacillus thuringiensis serovar israelensis based. The biological control uses the new method in 36.4% of the state area, assisting about 3,500,000 inhabitants.

Anesthesia of Biomphalaria spp. (Mollusca, Gastropoda): sodium pentobarbital is the drug of choice

The anesthetic effect of some water-soluble anesthesic or narcotic drugs currently used in mice was tested in molluscs of the Biomphalaria genus. Sodium thiopental was very toxic to the snails resulting in high rates of mortality in all the treatment schedules tested. Cetamine base, at concentration of 0.25 mg/ml of water, resulted in partial snail anesthesia (40% of snails were anesthetized) only after 20 h of exposition. The association of Cetamine base with Tiazine chloridrate did not improve the anesthesic effect, and higher concentrations of these drugs were toxic to the snails. Sodium pentobarbital at 0.4 mg/ml in water for 8 h was the best treatment schedule to anesthetize Biomphalaria snails. In this schedule, the snails were anesthetized without any toxic effect. The procedure provides a powerful tool for in vivo studies that demande a complete state of snail anesthesia.

In vitro interaction of Brazilian strains of the Nematode-trapping fungi Arthrobotrys spp. on Panagrellus sp. and Cooperia punctata

In vitro tests were carried out to verify the activity of 26 Brazilian isolates of predatory fungi of the genus Arthrobotrys on a free-living nematode (Panagrellus sp.) and on infective larvae of Cooperia punctata, a parasitic gastrointestinal nematode of cattle. The results showed that the free-living nematode Panagrellus sp. was the most preyed upon, compared to C. punctata, for all the fungal treatments. Also, variable predatory capacity was observed for different fungal isolates belonging to the same genus when applied to different nematode species.

The distribution of Biomphalaria spp. in different habitats in relation to physical, biological, water contact and cognitive factors in a rural area in Minas Gerais, Brazil

A total of 256 sites in 11 habitats were surveyed for Biomphalaria in Melquiades rural area (State of Minas Gerais) in August and November 1999 and in March 2000. Of the 1,780 Biomphalaria collected, 1,721 (96.7%) were B. glabrata and 59 (3.3%) B. straminea. Snails were found in all habitats except in wells, with the largest mean numbers in tanks, seepage ponds and canals, and the smallest numbers in springs, rice fields and fishponds. People's knowledge of the occurrence of Biomphalaria at the collection sites and the presence of Biomphalaria ova were strongly correlated with the occurrence of snails, and distance between houses and collection sites, as well as water velocity were inversely correlated with Biomphalaria occurrence (p < 0.001). The strongest predictor o f Biomphalaria occurrence was the presence of tilapia fish in fishponds. Fourteen Biomphalaria (0.8% of all snails) found at 6 sites were infected with Schistosoma mansoni. Suggestions are made for the utilization of local people's knowledge in snail surveys and further studies are recommended on the possible use of tilapia for biological control of Biomphalaria in fishponds, as well as modeling of S. mansoni transmission and reinfection.

Cotton Rats (Sigmodon hispidus) and Black Rats (Rattus rattus) as Possible Reservoirs of Leishmania spp. in Lara State, Venezuela

A total of 519 wild animals belonging to eleven species were collected during a two year study in a cutaneous leishmaniasis endemic area in Venezuela (La Matica, Lara State). The animals were captured in home-made Tomahawk-like traps baited with maize, bananas or other available local fruits, and parasites were isolated from 27 specimens. Two different species were found naturally infected with flagellates, i.e., cotton rats (Sigmodon hispidus) and black rats (Rattus rattus). Characterization of the parasites using PCR, kDNA restriction pattern and hybridization with species-specific probes revealed the presence of Leishmania (L.) mexicana in three of the black rats and Leishmania (V.) braziliensis in two others. The latter species was also identified in the single positive specimen of S. hispidus. The results suggested both species of animals as possible reservoirs of Leishmania sp.

Detection of hilA gene sequences in serovars of Salmonella enterica sufigbspecies enterica

hilA gene promoter, component of the Salmonella Pathogenicity Island 1, has been found in Salmonella serovar Typhimurium, being important for the regulation of type III secretion apparatus genes. We detected hilA gene sequences in Salmonella serovars Typhi, Enteritidis, Choleraesuis, Paratyphi A and B, and Pullorum, by polymerase chain reaction (PCR) and hybridization techniques. The primers to carry out PCR were designed according to hilA sequence. A low stringency hybridization with the probe pVV441 (hilA open-reading-frame plasmid) was carried out. To find hilA gene sequences in other Salmonella sp. suggest that these serovars could have similar sequences of this kind of virulence genes.

Morphologic and morphometric analysis of Hepatozoon spp. (Apicomplexa, Hepatozoidae) of snakes

Hepatozoon species are the most abundant hemoparasites of snakes. Its identification has been based mainly on the morphologic characterization of the gamonts in the peripheral blood of the vertebrate host and also of the cysts found in the internal organs of the vertebrate and invertebrate hosts. Using a computerized image analysis system, we studied five species of Hepatozoon from recently captured snakes in Botucatu, State of São Paulo, Brazil, to evaluate the importance of the morphology and morphometry of the gamonts for the characterization of Hepatozoon species and to analyze the morphologic changes induced in the erythrocytes by the parasite. The studied species were H. terzii of Boa constrictor amarali, Hepatozoon sp. of Crotalus durissusterrificus, H. philodryasi of Philodryas patagoniensis, and H. migonei and H. cyclagrasi of Hydrodynastes gigas. We observed three different groups, one of them including the species H. terzii, H. philodryasi and Hepatozoon sp. of C. durissus terrificus; and the other two consisting of H. migonei and H. cyclagrasi. Degree of alterations in the erythrocytes was variable and it may be useful for characterization of Hepatozoon species.

Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39) were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana) and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6%) presented a mixed infection Leishmania complex species, 17 (58.6%) a mixed infection Leishmania-T. cruzi, and 4 (13.8%) a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%). The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages) dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V.) braziliensis, L. (L.) chagasi and L. (L.) mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V.) braziliensis-L. (L.) mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.

Use of polymerase chain reaction and enzymatic cleavage in the identification of Helicobacter spp. in gastric mucosa of human beings from North Paraná, Brazil

Helicobacter pylori is the most common gastric bacteria of human beings. Animal-borne helicobacter have been associated with gastritis, ulceration, and gastric mucosa-associated lymphoid-tissue lymphoma in people. We attempted to identify the species of Helicobacter spp. that infect human beings in north Paraná, Brazil. Samples of gastric mucosa from 38 dyspeptic patients were analyzed by optic microscopy on silver stained slides, polimerase chain reaction (PCR), and enzymatic cleavage. Genus and species-specific primers to H. pylori, H. heilmannii, H. felis, and consensual primers to H. bizzozeronii or H. salomonis were used. The PCR products were submitted to enzymatic cleavage by VspI (Helicobacter spp. product) and HinfI (species products) enzymes. Thirty-two out of 38 patients evaluated had 3.2 to 5 µm long bacteria that resembled H. pylori in Warthin-Starry stained slides and were positive to the genus Helicobacter by PCR. In 30 of these patients the bacteria were identified as H. pylori. Two samples positive by silver stain were negative to all species tested by PCR. None of the 38 samples was positive to animal-origin helicobacter species. These results show that PCR and enzymatic restriction are practical methods to identify the species of helicobacters present in gastric mucosa of human beings. People in north Paraná appear to be infected mostly with H. pylori.

Species distribution and antifungal susceptibility profile of Candida spp. bloodstream isolates from Latin American hospitals

From March 1999 to March 2000, we conducted a prospective multicenter study of candidemia involving five tertiary care hospitals from four countries in Latin America. Yeast isolates were identified by classical methods and the antifungal susceptibility profile was determined according to the National Committee for Clinical Laboratory Standards microbroth assay method. During a 12 month-period we were able to collect a total of 103 bloodstream isolates of Candida spp. C. albicans was the most frequently isolated species accounting for 42% of all isolates. Non-albicans Candida species strains accounted for 58% of all episodes of candidemia and were mostly represented by C. tropicalis (24.2%) and C. parapsilosis (21.3%). It is noteworthy that we were able to identify two cases of C. lusitaniae from different institutions. In our casuistic, non-albicans Candida species isolates related to candidemic episodes were susceptible to fluconazole. Continuously surveillance programs are needed in order to identify possible changes in the species distribution and antifungal susceptibility patterns of yeasts that may occurs after increasing the use of azoles in Latin American hospitals.

The effect of nitric oxide combined with fluoroquinolones against Salmonella enterica serovar Typhimurium in vitro

Two regulons, soxRS and marRAB, are associated with resistance to quinolones or multiple antibiotic in Salmonella enterica serovar Typhimurium. These regulons are activated by nitric oxide and redox-cycling drugs, such as paraquat and cause on activation of the acrAB-encoded efflux pump. In this study, we investigated the effect of nitric oxide (NO) alone and in combination with ofloxacin, ciprofloxacin, and pefloxacin against S. typhimurium clinical isolates and mutant strains in vitro. We did not observe synergistic effect against clinical isolates and SH5014 (parent strain of acr mutant), while we found synergistic effect against PP120 (soxRS mutant) and SH7616 (an acr mutant) S. typhimurium for all quinolones. Our results suggest that the efficiencies of some antibiotics, including ofloxacin, ciprofloxacin, and pefloxacin are decreased via activation of soxRS and marRAB regulons by NO in S. enterica serovar Typhimurium. Further studies are warranted to establish the interaction of NO with the genes of Salmonella and, with multiple antibiotic resistance.