Louse infestation of the Chiribaya Culture, Southern Peru: variation in prevalence by age and sex

In order to improve the interpretive potential of archaeoparasitology, it is important to demonstrate that the epidemiology of ancient parasites is comparable to that of modern parasites. Once this is demonstrated, then we can be secure that the evidence of ancient parasitism truly reflects the pathoecology of parasitic disease. Presented here is an analysis of the paleoepidemiology of Pediculus humanus infestation from 146 mummies from the Chiribaya culture 1000-1250 AD of Southern Peru. The study demonstrates the modern parasitological axiom that 10% of the population harbors 70% of the parasites holds true for ancient louse infestation. This is the first demonstration of the paleoepidemiology of prehistoric lice infestation.

Changes associated with laboratory rearing in antennal sensilla patterns of Triatoma infestans, Rhodnius prolixus, and Rhodnius pallescens (Hemiptera, Reduviidae, Triatominae)

We examined changes in the array of antennal sensilla of three species of Triatominae (Triatoma infestans, Rhodnius prolixus, and R. pallescens) following their establishment for different periods in laboratory culture. In each case, the laboratory colonies were compared with conspecific samples taken directly from the field, by quantitative analysis of the sensilla arrays on the three distal segments of the antenna in terms of the densities of three types of chemoreceptors (basiconics and thick and thin walled trichoids) and one type of mechanoreceptor (bristles). Sensilla densities were compared by ANOVA or non-parametric tests, and by multivariate discriminant analysis. Strains of the same species reared in different laboratories showed significant differences in their sensilla arrays, especially when compared to field-collected material from the same geographic origin. A Bolivian strain of T. infestans reared in the laboratory for 15 years and fed at monthly intervals, showed greatest differences from its conspecific wild forms, especially in terms of reductions in the number of chemoreceptors. By contrast, an Argentine strain of T. infestans reared for 25 years in the laboratory and fed weekly, showed a relative increase in the density of mechanoreceptors. A Colombian strain of R. prolixus reared for 20 years and fed weekly or fortnightly, showed only modest differences in the sensilla array when compared to its wild populations from the same area. However, a Colombian strain of R. pallescens reared for 12 years and fed fortnightly, did show highly significant reductions in one form of chemoreceptor compared to its conspecific wild populations. For all populations, multivariate analysis clearly discriminated between laboratory and field collected specimens, suggesting that artificial rearing can lead to modifications in the sensory array. This not only supports the idea of morphological plasticity in these species, but also suggests caution in the use of long-established laboratory material for experimental studies designed to extrapolate the natural behaviour and physiology of these species.

Leishmania (Viannia) braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania) amazonensis

We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania) amazonensis replication rates were similar in both tissue culture media and reached maximum rates by 48 h. In contrast Leishmania (Viannia) braziliensis growth was significantly greater in TC-100 but maximum rates were achieved by 96 h. Folic acid appears to be the limiting factor and supplementation of Schneider media with this nutrient improved L. (V.) braziliensis replication rates and decreased the time of maximum replication to 48 h.

Suppression of Brugia malayi (sub-periodic) larval development in Aedes aegypti (Liverpool strain) fed on blood of animals immunized with microfilariae

Preliminary studies were carried out to investigate the role of filarial specific antibodies, raised in an animal model against the filarial parasite, Brugia malayi (sub-periodic), in blocking their early development in an experimental mosquito host, Aedes aegypti (Liverpool strain). In order to generate filarial specific antibodies, Mongolian gerbils, Meriones unguiculatus, were immunized either with live microfilariae (mf) of B. malayi or their homogenate. Mf were harvested from the peritoneal cavity of Mongolian gerbils with patent infection of B. malayi and fed to A. aegypti along with the blood from immunized animals. Development of the parasite in infected mosquitoes was monitored until they reached infective stage larvae (L3). Fewer number of parasites developed to first stage (L1) and subsequently to L2 and L3 in mosquitoes fed with blood of immunized animals, when compared to those fed with blood of control animals. The results thus indicated that filarial parasite specific antibodies present in the blood of the immunized animals resulted in the reduction of number of larvae of B. malayi developing in the mosquito host.

Primary culture of the region of the amebocyte-producing organ of the snail Biomphalaria glabrata, the intermediate host of Schistosoma mansoni

Biomphalaria glabrata snails are major hosts for the digenetic trematoda Schistosoma mansoni, the causative agent of human schistosomiasis. The success or failure of the infection will be dependent on the mobilization of the molluskan internal defense system, where a major role will be played by circulating hemocytes produced by the APO (amebocyte-producing organ) of the snail. In this report, the primary culture of the APO region of B. glabrata was obtained for the first time, as well as a control culture of the ovotestis. Three different cell populations migrated easily from the explants in culture, with no need of any dispersion agent. The cells grew in suspension at an incubation temperature of 15ºC and the cultures were maintained viable for up to two weeks. Two of these cell populations obtained resembled cell types known to be present in the hemolymph of Biomphalaria. The availability of APO cells in culture may contribute to a better understanding of the internal defense in mollusks, in general, as well as the specific response of B. glabrata to S. mansoni infection.

Biology of three species of the Meccus phyllosomus complex (Hemiptera: Reduviidae: Triatominae) fed on blood of hens and rabbits

Aspects related to hatching, life time, number of blood meals to molt, mortality, feeding time and postfeed defecation delay for each instar of Meccus phyllosomus, M. mazzottii, and M. bassolsae, life-cycle were evaluated and compared in two cohorts of each of those three species, fed on hens or rabbits. No significant (p > 0.05) differences were recorded among cohorts fed on hens respect to cohorts fed on rabbits in M. phyllosomus and M. mazzottii and the average time of hatching was 21.5 days for cohorts fed on hens and 22.5 for cohorts fed on rabbits. Average egg-to-adult development times were no significant (p > 0.05) different between both cohorts of M. phyllosomus and M. mazzotti, independent of the blood meal source. The average span in days for each instar fed on hens was not significantly different to the average span for each instar fed on rabbits, when comparisons were made by species. The number of blood meals at each nymphal instar varied from 1 to 6 in both cohorts of each species. The mortality rates were higher on older nymphs, in both cohorts of M. phyllosomus and M. bassolsae, whereas they were higher on first instar nymphs on M. mazzottii. Mean feeding time was no significant (p > 0.05) different in triatomines fed on hens or fed on rabbits, when each species were compared separately. A similar number of nymphs of each cohort, completed the cycle. Defecation delay was no significant (p > 0.05) different when cohorts fed on hens and fed on rabbits were compared by species. Most of the studied parameters showed no significant (p > 0.05) differences among those cohorts fed on hens and for fed on rabbits, which could mean a high degree of association of those species with birds as much as mammals, under wild conditions, increasing their capacity to colonize human dwellings.

Hepatic stereology of schistosomiasis mansoni infected-mice fed a high-fat diet

High-fat diets induce weight gain and fatty liver in wild-type mice. Schistosomiasis mansoni infection also promotes hepatic injury. This study was designed to quantify hepatic alterations in schistosomiasis mansoni-infected mice fed a high fat-rich chow compared to mice fed a standard rodent chow, using stereology. Female SW mice fed each either high-fat diet (29% lipids) or standard chow (12% lipids) over 8 months, and then were infected with Schistosoma mansoni cercariae. Four experimental groups were studied: infected mice fed a high-fat diet (IHFC) or standard chow (ISC), uninfected mice fed a high-fat diet (HFC) or standard chow (SC). Mice were sacrificed during early infection (9 weeks from exposure). The following hepatic biometry and the stereology parameters were determined: volume density (hepatocytes [h], sinusoids [s], steatosis [st] and hepatic fibrosis [hf]); numerical density (hepatocyte nuclei - Nv[h]); absolute number of total hepatocyte N[h], normal hepatocyte N[nh], and binucleated hepatocyte N[bh], percentage of normal hepatocyte P[nh] and binucleated hepatocyte P[bh]. IHFC and HFC groups exhibited TC, HDL-C, LDL-C, and body mass significantly greater (p < 0.05) than control group. No significant differences were found regards liver volume (p = 0.07). Significant differences were observed regards P[nh] (p = 0.0045), P[bh] (p = 0.0045), Nv[h] (p = 0.0006), N[h] (p = 0.0125), N[bh] (p = 0.0164) and N[nh] (p = 0.0078). IHFC mice group presented 29% of binucleated hepatocytes compared to HFC group (19%), ISC group (17%) and SC (6%). Volume density was significantly different between groups: Vv[h] (p = 0.0052), Vv[s] (p = 0.0025), Vv[st] (p = 0.0004), and Vv[hf] (p = 0.0007). In conclusion, schistosomiasis mansoni infection with concurrent high-fat diet promotes intensive quantitative changes in hepatic structure, contributing to an increasing on hepatic regeneration.

More productive in vitro culture of Cryptosporidium parvum for better study of the intra- and extracellular phases

The great difficulties in treating people and animals suffering from cryptosporidiosis have prompted the development of in vitro experimental models. Due to the models of in vitro culture, new extracellular stages of Cryptosporidium have been demonstrated. The development of these extracellular phases depends on the technique of in vitro culture and on the species and genotype of Cryptosporidium used. Here, we undertake the molecular characterization by polymerase chain reaction-restriction fragment lenght polymorphism of different Cryptosporidium isolates from calves, concluding that all are C. parvum of cattle genotype, although differing in the nucleotide at positions 472 and 498. Using these parasites, modified the in vitro culture technique for HCT-8 cells achieving greater multiplication of parasites. The HCT-8 cell cultures, for which the culture had not been renewed in seven days, were infected with C. parvum sporozoites in RPMI-1640 medium with 10% IFBS, CaCl2 and MgCl2 1 mM at pH 7.2. Percentages of cell parasitism were increased with respect to control cultures (71% at 48 h vs 14.5%), even after two weeks (47% vs 1.9%). Also, the percentage of extracellular stages augmented (25.3% vs 1.1% at 96 h). This new model of in vitro culture of C. parvum will enable easier study of the developmental phases of C. parvum in performing new chemotherapeutic assays.

Biology of three species of North American Triatominae (Hemiptera: Reduviidae: Triatominae) fed on rabbits

Aspects related to hatching, lifetime, number of blood meals for molting, mortality, feeding time, and postfeeding defecation delay were evaluated and compared in each instar of three North American Triatominae: Triatoma gerstaeckeri, Triatoma lecticularia and Triatoma protracta, all of them fed on rabbits. No significant differences (p > 0.05) were found among the three species regarding mean hatching rate, which was close to 20 days. Egg-to-adult development times were significantly shorter (p < 0.05) for T. lecticularia. Number of blood meals for molting to next instar ranged from one to five for T. protracta, and from one to six for T. gerstaeckeri and T. lecticularia. Mortality rates were higher in younger nymphs of T. lecticularia and T. protracta, while rates in T. gerstaeckeri were higher in fifth-instar nymphs. Mean feeding time was longest in T. gerstaeckeri, followed by T. lecticularia. More than twice the number of T. gerstaeckeri nymphs completed the development process, if compared to the nymphs from the other two species. Defecation delay was less than 10 min for T. lecticularia, T. protracta and the youngest nymphs of T. gerstaeckeri. Results point out that these three species may be important potential vectors of Trypanosoma cruzi for human populations, in areas of Mexico where these species are currently present.

Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.

Lesion aspirate culture for the diagnosis and isolation of Leishmania spp. from patients with cutaneous leishmaniasis

The detection of Leishmania spp. in skin lesion aspirates, using a puncture technique, was evaluated in 76 patients with cutaneous leishmaniasis (CL) who were referred to a Leishmaniasis Reference Centre in Brazil. CL was defined based on skin lesions suggestive of the disease and on a positive result of the Montenegro skin test or Giemsa-stained imprints of biopsy fragments. The aspirates were cultured using a vacuum tube device containing culture medium and evaluated for the presence of Leishmania spp. The biphasic medium culture was examined once a week for three weeks. Promastigotes were observed in 53/76 (69.7%) cultures. Stained smears from 60 of the 76 patients were evaluated using PCR-RFLP to detect the conserved minicircle region of Leishmania spp. and to classify the parasite. Of these patients, 45 (75%) showed positive results in aspirate culture and 15 presented negative results. The PCR was positive in 80% (53/60) samples. The PCR-RFLP profile was determined in 49 samples, of which 45 (92%) showed a pattern compatible with Leishmania (Viannia) braziliensis. The aspirate culture is a sensitive and feasible method for diagnosing CL and may be routinely adopted by health services for L. (V.) braziliensis isolation and identification.

Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies

The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.

Genus-specific kinetoplast-DNA PCR and parasite culture for the diagnosis of localised cutaneous leishmaniasis: applications for clinical trials under field conditions in Brazil

The positivities of two methods for the diagnosis of localised cutaneous leishmaniasis (CL) were estimated in 280 patients enrolled in a clinical trial. The trial was conducted in an endemic area of Leishmania (Viannia) braziliensis and trial participants were patients with skin ulcers and positive leishmanin skin tests. Patients underwent aspirative skin punctures of the ulcerated lesions and lymph nodes for in vitro cultures, which were processed under field conditions at the local health centre. Skin lesion biopsies were tested at a reference laboratory using kinetoplastid DNA (kDNA)-PCR to detect DNA. The median time required to obtain a positive culture from the skin samples was seven days and the contamination rate of the samples was 1.8%. The positivities of the cultures from skin lesions, kDNA-PCR and the combination of the two methods were 78.2% (95% CI: 73-82.6%), 89.3% (95% CI: 85.1-92.4%) and 97.1% (95% CI: 94.5-98.5%). We conclude that parasite culture is a feasible method for the detection of Leishmania in field conditions and that the combination of culture and PCR has a potential role for the diagnosis of CL in candidates for clinical trials.

Effect of untreated bed nets on blood-fed Phlebotomus argentipes in kala-azar endemic foci in Nepal and India

Observational studies in the Indian subcontinent have shown that untreated nets may be protective against visceral leishmaniasis (VL). In this study, we evaluated the effect of untreated nets on the blood feeding rates of Phlebotomus argentipes as well as the human blood index (HBI) in VL endemic villages in India and Nepal. The study had a "before and after intervention" design in 58 households in six clusters. The use of untreated nets reduced the blood feeding rate by 85% (95% CI 76.5-91.1%) and the HBI by 42.2% (95% CI 11.1-62.5%). These results provide circumstantial evidence that untreated nets may provide some degree of personal protection against sand fly bites.

Evaluation of buffy coat 16S rRNA PCR, buffy coat culture and whole blood PCR for detection of bacteraemia

The use of Gram type-specific PCR on buffy coat from clinical specimens for the detection of bacteraemia was evaluated for the first time using whole blood culture as the gold standard. In addition, the established buffy coat culture and whole blood PCR were also compared. Gram-positive bacteria belonging to six species and Gram-negative bacteria from 10 species were isolated and identified by culture and detected using broad-range 16S rDNA primers and Gram-specific primers. Data from the three methods all conferred very high sensitivity, specificity, positive and negative predictive values when compared to whole blood culture. The Kappa coefficients of agreement were 0.9819 (buffy coat PCR), 0.9458 (whole blood PCR) and 1.0 (buffy coat culture), which establishes their validity as alternative methods to routine blood culture in detecting bacteraemia. In addition, results showed that there was a direct correlation of WBC counts greater than 12,000 cells per mm³ to the occurrence of bacteraemia as detected by the four methods (p < 0.05).