Characterization of tabanid flies (Diptera: Tabanidae) in South Africa and Zambia and detection of protozoan parasites they are harbouring.

Tabanids are haematophagous flies feeding on livestock and wildlife. In the absence of information on the relationship of tabanid flies and protozoan parasites in South Africa and Zambia, the current study was aimed at characterizing tabanid flies collected in these two countries as well as detecting protozoan parasites they are harbouring. A total of 527 tabanid flies were collected whereby 70·2% were from South Africa and 29·8% were from Zambia. Morphological analysis revealed a total of five different genera collected from the sampled areas namely: Ancala, Atylotus, Haematopota, Philoliche and Tabanus. DNA extracted from South African Tabanus par and Tabanus taeniola tested positive for the presence of Trypanosoma congolense (Savannah) and Trypanosoma theileri whilst one member from T. par was positive for Trypanosoma brucei species. DNA extracted from Zambian tabanid flies tested positive for the presence of Besnoitia species at 1·27% (2/157), Babesia bigemina 5·73% (9/157), Theileria parva 30·11% (30/157) and 9·82% (14/157) for Trypanosoma evansi. This study is the first to report on relationship of Babesia and Theileria parasites with tabanid flies. Further investigations are required to determine the role of tabanids in transmission of the detected protozoan parasites in livestock and wildlife in South Africa and Zambia.

Quantitative analysis of cytokine mRNA expression and protozoan DNA load in Theileria parva-infected cattle.

Theileria parva (T. parva) causes a highly serious bovine disease called East Coast fever (ECF), which is characterized by pyrexia, dyspnea and cachexia and is of great economic importance in African countries. We hypothesize that the clinical symptoms of ECF could be explained by a cytokine dysregulation. In this study, we investigated the relationship between T. parva DNA load and expression levels of cytokine mRNAs in leukocytes from experimentally infected calves by quantitative PCR. The p104 gene, which encodes the T. parva 104 kDa microneme-rhoptry protein, was detected in cattle blood from day 10 after T. parva-infected tick infestation, and the protozoan DNA load was increased together with severity of disease. The mRNA expressions of pro-inflammatory cytokines, such as interleukin (IL)-1beta and IL-6, were up-regulated with protozoan DNA load increasing. In addition, the level of a type-2 cytokine (IL-10) transcript was also increased during the acute phase. In contrast, the down-regulation or no detectable levels of the expression of type-1 cytokines, such as IL-2 and interferon (IFN)-gamma were observed in T. parva-infected animals. Thus, our observations indicated that high protozoan load and resulting intense inflammatory responses might be involved in the severity of clinical signs observed in T. parva-infection.

Attachment duration required for Rhipicephalus appendiculatus to transmit Theileria parva to the host.

Theileria parva, the agent of East Coast fever (ECF), is transmitted to the host during the blood meal feeding of Rhipicephalus appendiculatus ticks. In order to investigate the relationship between the attachment duration of R. appendiculatus and the transmission of T. parva, infected adult ticks were allowed to attach to naive mice for variable lengths of time. Attached ticks and host animal's back skin biopsies from the tick attachment site were collected daily, starting from 24 hours post-tick attachment, and used for seminested polymerase chain reaction (PCR) detection of T. parva. T. parva-infected ticks started to transmit the parasites from 72 hours post-tick attachment. As expected, the transmission of T. parva from ticks to mouse skin increased with duration of tick attachment. Transmission of the parasites was 77.7%, 100%, 85.5%, and 100% on day 4, 5, 6, and 7 post-tick attachment, respectively, as could be detected from mice skin biopsies taken from T. parva-infected ticks' attachment sites. These results have important implications for our understanding of early events in the transmission of T. parva and would help in the development of effective pharmacologic substances and/or vaccines against ticks.

Direct detection of falciparum and non-falciparum malaria DNA from a drop of blood with high sensitivity by the dried-LAMP system.

BACKGROUND: Because of the low sensitivity of conventional rapid diagnostic tests (RDTs) for malaria infections, the actual prevalence of the diseases, especially those caused by non-Plasmodium falciparum (non-Pf) species, in asymptomatic populations remain less defined in countries lacking in well-equipped facilities for accurate diagnoses. Our direct blood dry LAMP system (CZC-LAMP) was applied to the diagnosis of malaria as simple, rapid and highly sensitive method as an alternative for conventional RDTs in malaria endemic areas where laboratory resources are limited. RESULTS: LAMP primer sets for mitochondria DNAs of Plasmodium falciparum (Pf) and human-infective species other than Pf (non-Pf; P. vivax, P. ovale, P. malariae) were designed and tested by using human blood DNA samples from 74 residents from a malaria endemic area in eastern Zambia. These malaria dry-LAMPs were optimized for field or point-of-care operations, and evaluated in the field at a malaria endemic area in Zambia with 96 human blood samples. To determine the sensitivities and specificities, results obtained by the on-site LAMP diagnosis were compared with those by the nested PCR and nucleotide sequencing of its product. The dry LAMPs showed the sensitivities of 89.7% for Pf and 85.7% for non-Pf, and the specificities of 97.2% for Pf and 100% for non-Pf, with purified blood DNA samples. The direct blood LAMP diagnostic methods, in which 1 µl of anticoagulated blood were used as the template, showed the sensitivities of 98.1% for Pf, 92.1% for non-Pf, and the specificities of 98.1% for Pf, 100% for non-Pf. The prevalences of P. falciparum, P. malariae and P. ovale in the surveyed area were 52.4, 25.3 and 10.6%, respectively, indicating high prevalence of asymptomatic carriers in endemic areas in Zambia. CONCLUSIONS: We have developed new field-applicable malaria diagnostic tests. The malaria CZC-LAMPs showed high sensitivity and specificity to both P. falciparum and non-P. falciparum. These malaria CZC-LAMPs provide new means for rapid, sensitive and reliable point-of-care diagnosis for low-density malaria infections, and are expected to help update current knowledge of malaria epidemiology, and can contribute to the elimination of malaria from endemic areas.

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

BACKGROUND: Loop-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. FINDINGS: The 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. CONCLUSIONS: Both 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.

PCR-based detection of blood parasites in cattle and adult Rhipicephalus appendiculatus ticks.

To ascertain the infection rate for tick-borne pathogens in Zambia, an epidemiological survey of Theileria parva, Babesia bigemina and Anaplasma marginale in traditionally managed Sanga cattle was conducted using PCR. Of the 71 native Zambian cattle, 28 (39.4%) were positive for T. parva, 16 (22.5%) for B. bigemina and 34 (47.9%) for A. marginale. The mixed infection rate in cattle was 8.5% (6/71), 16.9% (12/71), 7.0% (5/71) and 2.8% (2/71) for T. parva/B. bigemina, T. parva/A. marginale, B. bigemina/A. marginale and T. parva/B. bigemina/A. marginale, respectively. To predict the risk for transmission of tick-borne pathogens from ticks to cattle, a total of 74 Rhipicephalus appendiculatus ticks were collected from a location where cattle had been found positive for T. parva. Of the ticks collected, 10 (13.5%) were found to be PCR-positive for T. parva. The results suggest that the infection rate for tick-borne pathogens was relatively high in Sanga cattle and that adult R. appendiculatus ticks were highly infected with T. parva.