Alteration of free serum amino acids in voles infected with Trypanosoma brucei gambiense.

Free serum amino acid pools of field voles, Microtus montanus, were determined over a 24 hr period, and compared to values obtained from voles infected with Trypanosoma brucei gambiense. The majority of amino acids in the control animals demonstrated a diurnal variation, peaking predominantly during the dark portion of the photoperiod. This trend was not evident in the infected animals. In addition, infected voles possessed an apparent state of hypoaminoacidemia, with levels of threonine, serine, valine, isoleucine, leucine, tryosine, and tryptophan typically below uninfected values. Alanine and proline, in contrast, were markedly increased at certain time points. Tyrosine (reduced by approximately 50%) and tryptophan (reduced to levels below detection) underwent the most pronounced drop in trypanosome-infected animals, indicating the possibility of a related alteration in pools of derivative biogenic amines in other tissues. This suggests a role for the latter 2 amino acids in the neuropsychiatric syndromes of African trypanosomiasis.     

Inhibition of protein kinase activity from Trypanosoma cruzi and Trypanosoma gambiense by 3'-deoxyadenosine.

3'-Deoxydadenosine was found to be a potent inhibitor of nucleoside-stimulated protein kinase activity from culture forms of Trypanosoma cruzi and bloodstream forms of Trypanosoma gambiense. The type of inhibition by 3'-deoxyadenosine was competitive with respect to ATP. The inhibition constants for 3'-deoxyadenosine were determined to be 0.11mM and 0.085mM for the enzyme from T. cruzi and T. gambiense, respectively. The apparent Km value for ATP was 0.2mM for both enzymes. 2'-Deoxyadenosine was less effective as inhibitor of the protein kinase activity. The inhibition constants were calculated to be 0.8mM and 0.67mM, respectively.     

Elevated serum and hepatic tyrosine aminotransferase in voles chronically infected with Trypanosoma brucei gambiense.

Chronic infections of voles (Microtus montanus) with Trypanosoma brucei gambiense result in marked elevations in both serum and hepatic tyrosine aminotransferase. Serum enzyme levels correlate poorly with hepatic levels but well with parasitemia. Because the trypanosome itself contains high levels of this same enzyme, it is hypothesized that lysis of extravascular parasites, possibly due to agglutination by variant-specific antibody, releases parasite enzymes into host tissues, resulting in the observed increase in serum enzyme levels. The possible role of increased tyrosine transamination in the behavioral syndrome of trypanosome-infected voles is considered.     

Effect of hydroxystilbamidine on the kinetoplast of Trypanosoma gambiense in mice.

Hydroxystilbamidine (OHSA), an inhibitor of DNA synthesis, was shown to induce the dyskinetoplastic forms (akinetoplastic forms, AK forms) of Trypanosoma gambiense. The mode of appearance of the AK forms after injection of various doses of OHSA into infected mice was examined. The results suggested that production of the AK form is due to the selective inhibition of kinetoplast duplication of the drug without any effect on nuclear and cytoplasmic multiplication. When the parasites were treated with moderate doses of OHSA, segmenting forms without stainable kinetoplasts, were occasionally seen but attempts to establish a clone of akinetoplastic parasite were unsuccessful. Electron microscopy of parasites obtained after OHSA treatment showed not only irregular division of the kinetoplast but also the disorganization of kinetonucleus with disappearance of its envelope. Therefore, it was concluded that the AK forms were also produced by OHSA through disappearance of the kinetoplast.     

Effect of neocarzinostatin on the nucleus, kinetoplast and microtubules of Trypanosoma gambiense and Trypanosoma evansi.

The antibiotic neocarzinostatin (NCS) induces the anucleate form, not the dyskinetoplastic form, of Trypanosoma gambiense and Trypanosoma evansi. Light and electron microscopic studies indicated that production of the anucleate form is due to delay or inhibition of nuclear division. Excess pellicular microtubules are formed after treatment of trypanosomes with NCS, suggesting that in trypanosomes the microtubules replicate by induction, not by division. NCS also causes deformation of the axonemal and spindle microtubules. The K clone of T. evansi is more sensitive than the AK clone to the effects of NCS in inhibiting nuclear division and inducing the anucleate form.     

Preparation and study of an exoantigen of Histoplasma capsulatum for serological reactions.

The aim of this study was to determine the usefulness of a yeast-phase exo-antigen of Histoplasma capsulatum in standard serologic reactions. Three native strains of H.capsulatum which belong to Mycology Center collection were employed. They were maintained in their yeast-phase by weekly subcultures in 2% dextrose broth agar at 37 degrees C. After one week incubation yeast cells were suspended in distilled water containing thimerosal and phenylmethyl sulfonyl fluoride at a concentration of 1:5000. This suspension was left at room temperature for 72 h, then the supernatant was separated by centrifugation and it was lyophilized. Proteins and polysaccharides concentrations were determined. Immunodiffusion (ID) tests were carried out with an antigenic dilution containing 1.4 mg/ml of proteins. This exo-antigen was submitted to SDS-PAGE. Seven protein fractions were detected but only two of them showed antigenic activity against a pool of positive human sera; the molecular weights of these two proteins were 97 kDa and 66 kDa respectively. A metabolic antigen from the mycelial phase of H. capsulatum was used as control. A rabbit gammaglobulin anti-H. capsulatum was prepared and employed as positive control in serologic reactions. The antigenic capacity of ten batches of this exo-antigen was studied by ID and counterimmunoelectrophoresis (CIE) tests using serum samples of 20 hamsters experimentally infected by intracardiac inoculation of the yeast-phase of H. capsulatum. All tests presented positive results after three weeks of the infection. Fifty sera from patients suffering progressive histopasmosis were analyzed: ID, CIE and complement fixation (CF) tests were performed in all cases. HIV negative patients presented 7/7 (100%) positive reactions with the yeast-phase exoantigen and 5/7 (71.4%) with histoplasmin. In HIV positive patients CIE and CF were the most sensitive serologic tests, they gave positive results in 15/43 cases (34.8%) with the yeast-phase exo-antigen and in 7/43 cases (13.9%) with histoplasmin. Sera from 10 patients with paracoccidioidomycosis, aspergillosis and candidiasis respectively were studied by ID with the aim of detecting serologic cross reactions. No cross reaction was detected in these serum samples. This yeast-phase exo-antigen of H. capsulatum is more sensitive than and equally as specific as control histoplasmin.     

Immunological purification and partial characterization of variant-specific surface antigen messenger RNA of Trypanosoma brucei brucei.

Polyadenylated RNA isolated from total polyribosomes of two variable antigen types (VATs) of T. brucei brucei were shown to program the synthesis, in mRNA-dependant reticulocyte lysates, of a wide variety of polypeptides. After immunoprecipitation of these cell-free products with an homologous antiserum raised against purified variant-specific surface antigen (VSSA), a major electrophoretic band was apparent on fluorography. It was confirmed that this band corresponds to the variable antigen since only an excess of purified homologous antigen will provoke competition. The apparent molecular weight of the in vitro synthesized antigen is about 63,000 daltons. The VSSA mRNA has been found in membrane-bound polyribosomes and a 15 fold immunological purification of this mRNA has been obtained, using partially purified anti-VSSA IgG in conjunction with inactivated Staphylococcus aureus.     

A pilot study demonstrating the identification of Trypanosoma brucei gambiense and T. b. rhodesiense in vectors using a multiplexed high-resolution melt qPCR

Human African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 10⁴ tryps/mL. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l. and produces results in less than 2 hours, avoiding gel electrophoresis and subjective analysis. This method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.     

Isolation and purification of a preparation possessing leukocytosis-stimulating properties from pertussis bacteria]

A homogeneous protein LSF-2 preparation was extracted from the cultural fluid of Bordetella pertussis strains of the 1.0.3 serological type by means of precipitation with ammonium sulphate and electrofocussing; this preparation proved to produce a marked leukocytosis-stimulating and a weak toxic action of delayed type in experiments on animals. Intraperitoneal administration of 5 mug of the LSF-2 preparation caused a rise of leukocytosis in mice to 100,000 cells per 1 mm3, a delay in the gain in weight beginning from the 3rd day of the administration and a late death of the animals in 5% of cases. The LSF-2 preparation protected the mice in infection with a virulent pertussis strain No. 18323 in the amount of from 12 to 91%, depending on the immunizing dose; its ImD50 was equal to 2.0 -2.4 mug of protein. The results of investigations carried out permitted to assess the role of this substance in the formation of specific immunity in pertussis infection.     

Modelling to infer the role of animals in gambiense human African trypanosomiasis transmission and elimination in the DRC

Gambiense human African trypanosomiasis (gHAT) has been targeted for elimination of transmission (EoT) to humans by 2030. Whilst this ambitious goal is rapidly approaching, there remain fundamental questions about the presence of non-human animal transmission cycles and their potential role in slowing progress towards, or even preventing, EoT. In this study we focus on the country with the most gHAT disease burden, the Democratic Republic of Congo (DRC), and use mathematical modelling to assess whether animals may contribute to transmission in specific regions, and if so, how their presence could impact the likelihood and timing of EoT.By fitting two model variants—one with, and one without animal transmission—to the human case data from 2000–2016 we estimate model parameters for 158 endemic health zones of the DRC. We evaluate the statistical support for each model variant in each health zone and infer the contribution of animals to overall transmission and how this could impact predicted time to EoT.We conclude that there are 24/158 health zones where there is substantial to decisive statistical support for some animal transmission. However—even in these regions—we estimate that animals would be extremely unlikely to maintain transmission on their own. Animal transmission could hamper progress towards EoT in some settings, with projections under continuing interventions indicating that the number of health zones expected to achieve EoT by 2030 reduces from 68/158 to 61/158 if animal transmission is included in the model. With supplementary vector control (at a modest 60% tsetse reduction) added to medical screening and treatment interventions, the predicted number of health zones meeting the goal increases to 147/158 for the model including animal transmission. This is due to the impact of vector reduction on transmission to and from all hosts.