George W. Lubega

The emergence of Taenia solium cysticercosis in Eastern and Southern Africa as a serious agricultural problem and public health risk

Pig production has increased significantly in the Eastern and Southern Africa (ESA) region during the past decade, especially in rural, resource-poor, smallholder communities. Concurrent with the increase in smallholder pig keeping and pork consumption, there have been increasing reports of porcine cysticercosis in the ESA region. This article reviews the findings concerning the presence and impact of porcine cysticercosis in seven of the ESA countries. Most of the reported findings are based on surveys utilising lingual palpation and post-mortem examination, however, some also used serological assays. In Tanzania, community-based studies on porcine cysticercosis indicate a prevalence of 17.4% in the northern highlands district of Mbulu and a prevalence range of 5.1-16.9% in the southern highlands. In Kenya recent surveys in the southwestern part of the country where smallholder pig keeping is popular indicate that 10-14% of pigs are positive for cysticercosis by lingual examination. Uganda has the most pigs in Eastern Africa, most of which are kept under smallholder conditions. Preliminary surveys in 1998 and 1999 at slaughterhouses in Kampala indicated a prevalence of porcine cysticercosis between 0.12 and 1.2%, however, a rural survey in northern Uganda in 1999 indicated 34-45% of pigs slaughtered in selected villages were infected. Additionally, a new survey of 297 pigs slaughtered in Kampala in 2002 indicated that pigs from the central region of the country were negative for cysticercosis while 33.7% of the pigs coming from the rural Lira district in the north were positive. Interestingly 8 piglet foetuses removed from an infected slaughtered sow coming from Lira district were all found to harbour cysts of T. solium providing evidence of congenital transmission of porcine cysticercosis. In Mozambique, abattoir records indicate that porcine cysticercosis is present in all provinces of the country. A serological survey on pigs in rural Tete Province found 15% of pigs positive. In Zimbabwe, a retrospective study in official abattoirs around the country from 1994 to 2001 reported a mean prevalence of 0.34% which is in contrast to a post-mortem survey in 1999, which showed that the prevalence of porcine cysticercosis in rural west Zimbabwe where smallholder pig keeping is popular was 28.6%. In Zambia, abattoir records reported porcine cysticercosis in six of the nine provinces. Routine meat inspection of 1316 pigs at a slaughter slab in Lusaka showed that 20.6% of the pigs had cysticercosis whereas serological testing of 874 pigs at the same abattoir indicated that 56.6% were found to have circulating antigens of Taenia solium. Field surveys based on lingual palpation in Southern and Eastern Provinces of Zambia revealed prevalences of 8.2-28.4 and 5.2%, respectively. South Africa has the largest number of pigs in Southern Africa and cysticercosis has been recognised as a problem in the country for many decades. There is strong evidence supporting the high prevalence of neurocysticercosis infecting humans from resource-poor areas of the country where pigs are being raised under smallholder conditions. In spite of this community-based surveys on porcine cysticercosis have never been conducted in South Africa and the last slaughterhouse survey was conducted nearly 40 years ago. The prevalences of porcine cysticercosis found in these ESA countries rank among the highest in the world and the disease is emerging as an important constraint for the nutritional and economic well being of resource-poor smallholder farming communities. The current findings suggest the widespread presence of human tapeworm carriers and thus a high risk of human cysticercosis in both rural areas and urban centres in the ESA region. More research is required in the region to assess the extent and public health and economic impact of T. solium infection in order to determine whether and what prevention and control efforts are needed.

Nucleic Acid Sequence-Based Amplification with Oligochromatography for Detection of Trypanosoma brucei in Clinical Samples

Molecular tools, such as real-time nucleic acid sequence-based amplification (NASBA) and PCR, have been developed to detect Trypanosoma brucei parasites in blood for the diagnosis of human African trypanosomiasis (HAT). Despite good sensitivity, these techniques are not implemented in HAT control programs due to the high cost of the equipment, which is unaffordable for laboratories in developing countries where HAT is endemic. In this study, a simplified technique, oligochromatography (OC), was developed for the detection of amplification products of T. brucei 18S rRNA by NASBA. The T. brucei NASBA-OC test has analytical sensitivities of 1 to 10 parasites/ml on nucleic acids extracted from parasite culture and 10 parasites/ml on spiked blood. The test showed no reaction with nontarget pathogens or with blood from healthy controls. Compared to the composite standard applied in the present study, i.e., parasitological confirmation of a HAT case by direct microscopy or by microscopy after concentration of parasites using either a microhematocrit centrifugation technique or a mini-anion-exchange centrifugation technique, NASBA-OC on blood samples had a sensitivity of 73.0% (95% confidence interval, 60 to 83%), while standard expert microscopy had a sensitivity of 57.1% (95% confidence interval, 44 to 69%). On cerebrospinal fluid samples, NASBA-OC had a sensitivity of 88.2% (95% confidence interval, 75 to 95%) and standard microscopy had a sensitivity of 86.2% (95% confidence interval, 64 to 88%). The T. brucei NASBA-OC test developed in this study can be employed in field laboratories, because it does not require a thermocycler; a simple heat block or a water bath maintained at two different temperatures is sufficient for amplification.

Immunization with a tubulin-rich preparation from Trypanosoma brucei confers broad protection against African trypanosomosis.

Tubulin from Trypanosoma brucei was purified to near homogeneity using a protocol which involved treatment with urea with subsequent renaturation and was then used to immunize mice. Renatured tubulin further purified by SDS-PAGE (denatured), synthetic tubulin peptides (STP), and rat brain tubulin (RbTub) were also used. Immunized mice were challenged with T. brucei, Trypanosoma congolense or Trypanosoma rhodesiense. Renatured T. brucei tubulin (nTbTub) induced protection in all mice tested, of which 60-80% (n = 81) was complete and the remainder partial. Denatured T. brucei tubulin (dTbTub), STP, or RbTub induced lower antibody levels than nTbTub and did not offer protection. However, in culture, the antibodies against dTbTub or STP killed trypanosomes although at lower dilutions than nTbTub, but those against RbTub did not. In Western blots anti-trypanosome antibodies recognized the tubulin of all the trypanosome species investigated but not vertebrate tubulin, whereas the anti-RbTUB antibodies recognized both trypanosome and vertebrate tubulin. Of the five mice given passive immunity by the transfer of anti-nTbTub serum, four were completely protected and one partially protected. These data suggest that tubulin is the relevant immunogen in the preparation used and could therefore be a promising target for the development of a parasite-specific, broad spectrum vaccine.

Drug-resistance of Trypanosoma b. rhodesiense isolates from Tanzania

Objective  To determine the drug resistance of Trypanosoma brucei rhodesiense strains isolated from sleeping sickness patients in Tanzania.

Theileria parva genetic diversity and haemoparasite prevalence in cattle and wildlife in and around Lake Mburo National Park in Uganda.

Wildlife, especially Cape buffalo (Syncerus caffer), are thought to act as a reservoir for many of the important tick-borne pathogens of cattle. In this study, we have determined the prevalence of the most significant tick-borne haemoparasites in wildlife (buffalo, impala, eland and bushbuck) as well as in cattle grazing inside and neighbouring Lake Mburo National Park (LMNP) in Uganda. A high percentage of buffalo were carriers of Theileria parva, Theileria mutans, Theileria velifera, Theileria buffeli and Theileria sp. (buffalo) as well as Anaplasma marginale and Anaplasma centrale. The majority of impala sampled were carriers of A. centrale, and all were carriers of an unidentified Babesia/Theileria species. The eland and bushbuck sampled were all carriers of Theileria taurotragi and Theileria buffeli, and the majority were carriers of T. mutans. The bushbuck sampled were also carriers for Erhlichia bovis. There were some differences in the prevalence of haemoparasites between the calves sampled inside and neighbouring LMNP. In order to address the question of whether there is evidence for interbreeding between buffalo-associated and cattle-associated T. parva populations, multi-locus genotypes (MLGs) of T. parva (based on micro-satellite markers) from buffalo and from calves grazing inside and outside LMNP were compared, and the results revealed that buffalo and cattle gene pools were distinct, showing no evidence for transmission of buffalo-derived T. parva genotypes to the cattle population.

Susceptibility of Ugandan Trypanosoma brucei rhodesiense isolated from man and animal reservoirs to diminazene, isometamidium and melarsoprol

Thirty‐six Trypanosoma brucei spp. stocks isolated from man and domestic animals in south‐east Uganda were studied for susceptibility to diminazene, isometamidium and melarsoprol in vitro. All stocks were susceptible to melarsoprol. One T. b. rhodesiense stock isolated from a sleeping sickness patient showed reduced susceptibility to the veterinary drugs diminazene and isometamidium. More than 100 ng/ml diminazene or 0.78 ng/ml isometamidium were required to eliminate that stock during 10 days drug exposure. In contrast, the remaining stocks were eliminated by 0.8–6.3 ng/ml diminazene and 0.01–0.20 ng/ml isometamidium. Clones derived from the resistant T. b. rhodesiense stock showed reduced susceptibility to isometamidium and diminazene comparable to the parental population. Control of sleeping sickness by treatment of the animal reservoir could, therefore, face serious problems since drug‐resistant stocks as reported here would most likely not be eliminated by recommended doses of diminazene and isometamidium.

Trypanosoma brucei: anti-tubulin antibodies specifically inhibit trypanosome growth in culture.

We previously observed that trypanosome tubulin immunizes mice against infection by this parasite. Here we describe the direct effect of anti-tubulin antibodies on trypanosomes, using rabbit antibodies to renatured (nTbTub) or SDS-PAGE denatured (dTbTub) Trypanosoma brucei tubulin. We also evaluate antibodies to synthetic tubulin peptides (STP) and rat brain tubulin (RbTub). The anti-nTbTub serum strongly inhibited trypanosome proliferation in culture, and immunoagglutinated trypanosomes even after heat inactivation of complement. The anti-dTbTub and the anti-STP sera also inhibited trypanosome growth and immunoagglutinated trypanosomes, but to a lesser extent than the anti-nTbTub, whereas the anti-RbTub serum had no effect. In Western blots these antibodies were species specific. Immunofluorescence showed that the surface of intact trypanosomes was not uniformly stained by any of these antibodies, but cells that had been permeabilised were labeled throughout the cytoplasm. This suggests that the variant surface glycoproteins (VSG) played no part in the generation of these inhibitory antibodies.

Detection of Trypanosoma brucei parasites in blood samples using real-time nucleic acid sequence-based amplification

Currently, the conventional diagnosis of human African trypanosomiasis (HAT) is by microscopic demonstration of trypomastigotes in blood, lymph, and/or cerebrospinal fluid. However, microscopic diagnosis of HAT is not sensitive enough and may give false-negative results, thus, denying the patient the necessary treatment of the otherwise fatal disease. For this reason, a highly sensitive technique needs to be developed to enhance case findings. In this study, the real-time nucleic acid sequence-based amplification assay described is based on amplification and concurrent detection of small subunit rRNA (18S rRNA) of Trypanosoma brucei. The sensitivity of the assay was evaluated on nucleic acid from in vitro cultured parasites and blood spiked with various parasites quantities. The assay detected 10 parasites/mL using cultured parasites as well as spiked blood. A sensitive assay such as the one developed in this study may become an alternative tool to confirm diagnosis of human African trypanosomiasis.

Haemoparasite prevalence and Theileria parva strain diversity in Cape buffalo (Syncerus caffer) in Uganda

Cape buffalo (Syncerus caffer) are considered to be an important reservoir for various tick-borne haemoparasites of veterinary importance. In this study we have compared the haemoparasite carrier prevalence in buffalo from four geographically isolated national parks in Uganda [Lake Mburo National Park (LMNP), Queen Elizabeth National Park (QENP), Murchison Falls National Park (MFNP) and Kidepo Valley National Park (KVNP)]. Differences were seen in haemoparasite prevalence in buffalo from the four national parks. All the buffalo sampled in LMNP were carriers of Theileria parva however, buffalo from MFNP and KVNP, which are both located in the north of Uganda, were negative for T. parva. Interestingly, 95% of buffalo in the northern part of QENP were T. parva positive, however all buffalo sampled in the south of the park were negative. A high multiplicity of infection was recorded in all the buffalo found to be carrying T. parva, with evidence of at least nine parasite genotypes in some animals. Most of the buffalo sampled in all four national parks were carriers of T. mutans and T. velifera, however none were carriers of T. taurotragi, Babesia bovis, Babesia bigemina, Ehrlichia bovis or Ehrlichia ruminantium. All the buffalo sampled from LMNP were positive for T. buffeli and T. sp. (buffalo) however, buffalo from the parks in the north of the country (KVNP and MFNP) were negative for these haemoparasites. Anaplasma centrale and Anaplasma marginale were circulating in buffalo from all four national parks. T. parva gene pools from two geographically separated populations of buffalo in two of the national parks in Uganda (LMNP and QENP) were compared. The T. parva populations in the two national parks were distinct, indicating that there was limited gene flow between the populations. The results presented highlight the complexity of tick-borne pathogen infections in buffalo and the significant role that buffalo may play as reservoir hosts for veterinary haemoparasites that have the potential to cause severe disease in domestic cattle.

Phase II evaluation of sensitivity and specificity of PCR and NASBA followed by oligochromatography for diagnosis of human African trypanosomiasis in clinical samples from D.R. Congo and Uganda.

Background The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) have been recently modified by coupling to oligochromatography (OC) for easy and fast visualisation of products. In this study we evaluate the sensitivity and specificity of the PCR-OC and NASBA-OC for diagnosis of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense human African trypanosomiasis (HAT). Methodology and Results Both tests were evaluated in a case-control design on 143 HAT patients and 187 endemic controls from the Democratic Republic of Congo (DRC) and Uganda. The overall sensitivity of PCR-OC was 81.8% and the specificity was 96.8%. The PCR-OC showed a sensitivity and specificity of 82.4% and 99.2% on the specimens from DRC and 81.3% and 92.3% on those from Uganda. NASBA-OC yielded an overall sensitivity of 90.2%, and a specificity of 98.9%. The sensitivity and specificity of NASBA-OC on the specimens from DRC was 97.1% and 99.2%, respectively. On the specimens from Uganda we observed a sensitivity of 84.0% and a specificity of 98.5%. Conclusions/Significance The tests showed good sensitivity and specificity for the T. b. gambiense HAT in DRC but rather a low sensitivity for T. b. rhodesiense HAT in Uganda.