Boniface Namangala

Relative Contribution of Interferon-γ and Interleukin-10 to Resistance to Murine African Trypanosomosis

Resistance to Trypanosoma brucei brucei has been correlated with the ability of infected animals to produce interferon (IFN)-gamma and tumor necrosis factor (TNF) in an early phase of infection, followed by interleukin (IL)-4 and IL-10 in late and chronic stages of the disease. Contributions of IFN-gamma and IL-10 in the control of parasitemia and survival of mice infected with T. brucei brucei were investigated by using IFN-gamma(-/-) and IL-10(-/-) mice. Results suggest that IFN-gamma, mainly secreted by CD8(+) T cells, is essential for parasite control via macrophage activation, which results in TNF and nitric oxide secretions. IL-10, partially secreted by CD4(+) T cells, seems to be important for the survival of infected mice. Its absence resulted in the sustained secretion of inflammatory mediators, which indicated the role of IL-10 in maintaining the balance between pathogenic and protective immune responses during African trypanosomosis.

Alternative versus classical macrophage activation during experimental African trypanosomosis

The type I/type II cytokine balance may influence the development of different subsets of suppressive macrophages, i.e., classically activated macrophages (caMphi, type I) versus alternatively activated macrophages (aaMphi, type II). Recently, we showed that although mice infected with phospholipase C-deficient (PLC-/-) Trypanosoma brucei brucei exhibit a clear shift from type I to the type II cytokine production, wild type (WT)-infected mice remain locked in a type I cytokine response. In the present study, phenotype and accessory cell function of macrophages elicited during WT and PLC-/- T. b. brucei infection were compared. Results indicate that caMphi develop in a type I cytokine environment in the early phase of WT and PLC-/- trypanosome infection, correlating with inhibition of T cell activation triggered by a mitogen, a superantigen, or an antigen. In the late stage of infection, only PLC(-/-)-infected mice resisting the infection develop type II cytokine-associated aaMphi correlating with impaired antigen- but not mitogen- or superantigen-induced T cell activation.

Infection Stage-Dependent Modulation of Macrophage Activation in Trypanosoma congolense-Resistant and -Susceptible Mice

ABSTRACT The contribution of cytokines and chemokines to resistance and susceptibility to African trypanosomiasis remains controversial. In the present study, the levels of type I and type II cytokines and of the MCP-1 chemokine were compared during the early and late stages of Trypanosoma congolense infection in susceptible BALB/c and resistant C57BL/6 mice. Moreover, the status of macrophage activation was compared in these animals by analyzing the inducible nitric oxide synthase-arginase balance, tumor necrosis factor secretion, and expression of the FIZZ1 and YM genes. Data show that changing from a predominant type I cytokine environment in the early stage of infection to a predominant type II cytokine environment and an enhanced MCP-1 secretion in the late stage of infection correlates with resistance to T. congolense. Concomitantly, macrophage activation evolves from a classical to a predominant alternative phenotype. We further confirmed that the simultaneous occurrence of type I/type II cytokines in the early stage of infection in susceptible BALB/c mice, reflected by the presence of macrophages exhibiting a mixed classical/alternative activation phenotype, is associated with uncontrolled parasite growth and early death. Interleukin-4 (IL-4) and IL-13 signaling did not influence the susceptibility of BALB/c mice to T. congolense infection and interestingly were not the main trigger to alternative macrophage activation. In T. congolense-resistant C57BL/6 mice, our results corroborated the induction of FIZZ1 and YM gene expressions with the alternative pathway of macrophage activation. In susceptible BALB/c mice, however, YM but not FIZZ1 induction reflected the emergence of alternatively activated macrophages. Hence, the FIZZ1 and YM genes may be useful markers to discriminate between distinct populations of alternatively activated macrophages.

Development of Loop-Mediated Isothermal Amplification (LAMP) assays for rapid detection of Ehrlichia ruminantium

BackgroundThe rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of E. ruminantium.ResultsTwo sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries.ConclusionsDue to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.

Seroepidemiological Prevalence of Multiple Species of Filoviruses in Fruit Bats (Eidolon helvum) Migrating in Africa

Fruit bats are suspected to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Using an enzyme-linked immunosorbent assay based on the viral glycoprotein antigens, we detected filovirus-specific immunoglobulin G antibodies in 71 of 748 serum samples collected from migratory fruit bats (Eidolon helvum) in Zambia during 2006-2013. Although antibodies to African filoviruses (eg, Zaire ebolavirus) were most prevalent, some serum samples showed distinct specificity for Reston ebolavirus, which that has thus far been found only in Asia. Interestingly, the transition of filovirus species causing outbreaks in Central and West Africa during 2005-2014 seemed to be synchronized with the change of the serologically dominant virus species in these bats. These data suggest the introduction of multiple species of filoviruses in the migratory bat population and point to the need for continued surveillance of filovirus infection of wild animals in sub-Saharan Africa, including hitherto nonendemic countries.

High prevalence of drug resistance in animal trypanosomes without a history of drug exposure.

Background Trypanosomosis caused by Trypanosoma congolense is a major constraint to animal health in sub-Saharan Africa. Unfortunately, the treatment of the disease is impaired by the spread of drug resistance. Resistance to diminazene aceturate (DA) in T. congolense is linked to a mutation modifying the functioning of a P2-type purine-transporter responsible for the uptake of the drug. Our objective was to verify if the mutation was linked or not to drug pressure. Methodology/Principal Findings Thirty-four T. congolense isolates sampled from tsetse or wildlife were screened for the DA-resistance linked mutation using DpnII-PCR-RFLP. The results showed 1 sensitive, 12 resistant and 21 mixed DpnII-PCR-RFLP profiles. This suggests that the mutation is present on at least one allele of each of the 33 isolates. For twelve of the isolates, a standard screening method in mice was used by (i) microscopic examination, (ii) trypanosome-specific 18S-PCR after 2 months of observation and (iii) weekly trypanosome-specific 18S-PCR for 8 weeks. The results showed that all mice remained microscopically trypanosome-positive after treatment with 5 mg/kg DA. With 10 and 20 mg/kg, 8.3% (n = 72) and 0% (n = 72) of the mice became parasitologically positive after treatment. However, in these latter groups the trypanosome-specific 18S-PCR indicated a higher degree of trypanosome-positivity, i.e., with a unique test, 51.4% (n = 72) and 38.9% (n = 72) and with the weekly tests 79.2% (n = 24) and 66.7% (n = 24) for 10 and 20 mg/kg respectively. Conclusion/Significance The widespread presence of the DA-resistance linked mutation in T. congolense isolated from wildlife suggests that this mutation is favourable to parasite survival and/or its dissemination in the host population independent from the presence of drug. After treatment with DA, those T. congolense isolates cause persisting low parasitaemias even after complete elimination of the drug and with little impact on the hosts health.

Direct blood dry LAMP: a rapid, stable, and easy diagnostic tool for Human African Trypanosomiasis.

Loop-mediated isothermal amplification (LAMP) is a rapid and sensitive tool used for the diagnosis of a variety of infectious diseases. One of the advantages of this method over the polymerase chain reaction is that DNA amplification occurs at a constant temperature, usually between 60–65°C; therefore, expensive devices are unnecessary for this step. However, LAMP still requires complicated sample preparation steps and a well-equipped laboratory to produce reliable and reproducible results, which limits its use in resource-poor laboratories in most developing countries. In this study, we made several substantial modifications to the technique to carry out on-site diagnosis of Human African Trypanosomiasis (HAT) in remote areas using LAMP. The first essential improvement was that LAMP reagents were dried and stabilized in a single tube by incorporating trehalose as a cryoprotectant to prolong shelf life at ambient temperature. The second technical improvement was achieved by simplifying the sample preparation step so that DNA or RNA could be amplified directly from detergent-lysed blood samples. With these modifications, diagnosis of HAT in local clinics or villages in endemic areas becomes a reality, which could greatly impact on the application of diagnosis not only for HAT but also for other tropical diseases.

Prevalence of Trypanosoma sp. in cattle from Tanzania estimated by conventional PCR and loop-mediated isothermal amplification (LAMP)

This study compared the prevalence of trypanosome infections estimated by PFR-loop-mediated isothermal amplification (LAMP) with conventional polymerase chain reaction (PCR) tests. One hundred forty eight cattle blood samples were collected from Robanda village, Mara region, Tanzania in April 2008. In conventional PCR, four sets of primers, specific for the detection of Trypanosoma sp., Trypanosoma brucei rhodesiense, Trypanosoma vivax, and Trypanozoon, as well as a modified LAMP were used. Conventional PCR detected no infection or up to 8, 1, and 3 infections with Trypanosoma congolense savannah, Trypanozoon, and T. vivax, respectively, whereas LAMP detected additional 44 Trypanozoon positive cases. Our results clearly indicate that the prevalence of Trypanozoon spp. in cattle in Robanda village estimated by PFR-LAMP (30.4%) was significantly higher than the estimates by PCR assays (0.6–2%). As such, future studies should target epidemiological surveys of Trypanozoon and T. brucei rhodesiense infections in possible reservoir animals by LAMP to further elucidate the actual prevalence of these parasites.

The use of Loop-mediated Isothermal Amplification (LAMP) to detect the re-emerging Human African Trypanosomiasis (HAT) in the Luangwa and Zambezi valleys

BackgroundLoop-mediated isothermal amplification (LAMP) is a novel strategy which amplifies DNA with high sensitivity and rapidity under isothermal conditions. In the present study, the performance of the repetitive insertion mobile element (RIME)-LAMP and human serum resistance-associated gene (SRA)-LAMP assays were evaluated using clinical specimens obtained from four male patients from Luangwa and Zambezi valleys in Zambia and Zimbabwe, respectively.FindingsThe cases reported in this preliminary communication were all first diagnosed by microscopy, through passive surveillance, and confirmed by both RIME-LAMP and SRA-LAMP. A good correlation between microscopy and LAMP was observed and contributed to staging and successful treatment of patient. RIME-LAMP and SRA-LAMP complimented each other well in all the cases.ConclusionsBoth RIME-LAMP and SRA-LAMP were able to detect Trypanosoma brucei rhodesiense DNA in patient blood and CSF and hence confirmed HAT in the parasitaemic patients. Our study indicates that the LAMP technique is a potential tool for HAT diagnosis, staging and may be useful for making therapeutic decisions. However, no statistically significant conclusion may be drawn due to the limited sample size used in the present study. It is thus imperative to conduct a detailed study to further evaluate the potential of LAMP as a bedside diagnostic test for HAT.

Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries.