High infection rate of tick-borne protozoan and rickettsial pathogens of cattle in Malawi and the development of a multiplex PCR for Babesia and Theileria species identification.

Malawi has an estimated cattle population of 1,884,803 heads, the indigenous Malawi zebu breed accounts for 91.2%, while the exotic and crossbred accounts for the remaining 8.8%. Although ticks and tick-borne diseases are widespread in Malawi, no molecular study has been conducted to investigate the tick-borne Anaplasmataceae and piroplasms infecting cattle. To provide an insight into the current status of tick-borne pathogens (TBPs) of cattle, a molecular survey was conducted in the central and southern regions of Malawi. A total of 191 cattle of which 132 were Malawi zebu, 44 were Holstein Friesian and 15 were Holstein-Friesian/ Malawi zebu crosses were screened for Anaplasmataceae and piroplasms using the heat shock protein groEL gene and 18S rDNA, respectively. A new 18S rDNA multiplex PCR assay was designed for Babesia and Theileria species identification without sequencing. Overall, 92.3% (n = 177) of the examined animals were infected with at least one TBP. Anaplasmataceae-positive rate was 57.6% (n = 110) while for piroplasms it was 80.1% (n = 153). The detected Anaplasmataceae were Anaplasma bovis 2.6% (n = 5), Anaplasma marginale 24.6% (n = 47), Anaplasma platys-like 13.6% (n = 26), uncharacterized Anaplasma sp. 14.1% (n = 27), and uncharacterized Ehrlichia sp. 16.2% (n = 31). The detected piroplasms were Babesia bigemina 2.6% (n = 5), Theileria mutans 73.8% (n = 141), Theileria parva 33.0% (n = 63), Theileria taurotragi 12.6% (n = 24), and Theileria velifera 53.4% (n = 102). Mixed infection rate was found in 79.6% (n = 152) of the samples analyzed. This study has shown a high burden of TBPs among cattle in Malawi which highlights the need to conceive new methods to control ticks and TBPs in order to improve animal health and productivity. The newly developed multiplex PCR assay would be a useful tool especially in resource limited settings where sequencing is not available and when mixed infections are expected.

Molecular identification and genetic characterization of tick-borne pathogens in sheep and goats at two farms in the central and southern regions of Malawi.

Tick-borne diseases (TBDs) caused by pathogens belonging to the genera Anaplasma, Ehrlichia, Babesia and Theileria in small ruminants are widespread in the tropical and sub-tropical countries. The epidemiology of tick-borne pathogens (TBPs) in small ruminants is less understood compared to those infecting cattle in general. This study was carried out to investigate and characterize TBPs in sheep and goats using molecular tools. A total of 107 blood samples from sheep (n = 8) and goats (n = 99) were collected from animals that were apparently healthy from two farms in the central and the southern regions of Malawi. The V4 hypervariable region of the 18S ribosomal RNA gene (rDNA) and the V1 hypervariable region of the 16S rDNA polymerase chain reaction (PCR) assays were used for detection of tick-borne piroplasms and Anaplasmataceae, respectively. Almost the full-length 18S rDNA and the heat shock protein (groEL) gene sequences were used for genetic characterization of the piroplasms and Anaplasmataceae, respectively. The results showed that 76.6 % of the examined animals (n = 107) were positive for at least one TBP. The overall co-infection with at least two TBPs was observed in forty-eight animals (45 %). The detected TBPs were Anaplasma ovis (65 %), Ehrlichia ruminantium (4%), Ehrlichia canis (2%), Babesia strain closely related to Babesia gibsoni (1%), Theileria ovis (52 %), Theileria mutans (3%), Theileria separata (2%), Anaplasma sp. (1%) and Theileria sp. strain MSD-like (17 %). To the authors knowledge this is the first molecular study of TBPs in sheep and goats in Malawi. These results have therefore provided a significant milestone in the knowledge of occurrence of TBPs in sheep and goats in Malawi, which is prerequisite to proper diagnosis and control.

The use of molecular technology to investigate trypanosome infections in tsetse flies at Liwonde Wild Life Reserve.

Background: Trypanosomes are protozoan flagellates that cause human African trypanosomiasis (HAT) and African animal trypanosomiasis (AAT). HAT is caused by Trypanosoma brucei rhodesiense in East and Central Africa and T.b. gambiense in West Africa, whereas AAT is caused by a number of trypanosome species, including T. brucei brucei, T. evansi, T. vivax, T. congolense, T. godfreyi and T. simiae. The aim of this study was to establish if tsetse flies at Liwonde Wild Life Reserve (LWLR) are infected with these trypanosomes and thus pose a risk to both humans and animals within and surrounding the LWLR. Methods: A total of 150 tsetse flies were caught. Of these, 82 remained alive after capture and were dissected such that the mid-gut could be examined microscopically for trypanosomes. DNA extractions were performed from both mid-guts and the 68 dead flies using a Qiagen Kit. Amplification techniques involved the Internal Transcriber Spacer 1 (ITS 1) conventional polymerase chain reaction (PCR) with primers designed to identify trypanosome species, and Repetitive Insertion Mobile Element - Loop Mediated Isothermal Amplification (RIME LAMP), a sequence specific to T. brucei. Results: Analysis showed that 79/82 (96.3%) of the mid-guts examined microscopically were positive for trypanosomes and that 75/150 (50%) of the DNA extracts (from the mid-gut, and tsetse fly carcasses) were positive for T. brucei, as determined by the RIME LAMP method. ITS1 PCR further showed that 87/150 (58.0%) flies were positive for trypanosomes, of which 56/87 (64.4%) were T. brucei, 9/87 (10.3%) were T. vivax; 7/87 (8.1%) were T. simiae; 6/87 (6.9%) were T. congolense, and 6/87 (6.9%) were T. godfreyi. Ten samples had a mixture of infections. Conclusion: Our analysis demonstrated a mixture of infections from trypanosome species in tsetse flies at LWLR, and that T. brucei, the species that causes HAT, was the most common. Our study successfully used molecular techniques to demonstrate the presence of T. b. rhodesiense at LWLR, a species that causes HAT in both East and Central Africa.

Comparative evaluation of dry and liquid RIME LAMP in detecting trypanosomes in dead tsetse flies.

Xenomonitoring is an important approach in assessing the progress of trypanosomiasis control as well as in estimating the endemicity of trypanosomes in affected areas. One of the major challenges in this approach is the unavailability of sensitive and easy to use xenomonitoring tools that can be used in the remote areas where the disease occurs. One tool that has been used successfully in detecting the parasites in tsetse flies is the repetitive insertion mobile element loop-mediated isothermal amplification (RIME LAMP). This tool has recently been modified from the liquid form to dry form for use in remote areas; however, uptake for use in the field has been slow. Field-collected tsetse flies were used to evaluate the performance of dry RIME LAMP over the conventional liquid RIME LAMP. All the samples were also subjected to internal transcribed spacer 1 (ITS1) ribosomal deoxyribonucleic acid (DNA) polymerase chain reaction (PCR) as a standard. ITS1-PCR-positive samples were further sequenced for confirmation of the species. A total of 86 wild tsetse flies were left to dry at room temperature for 3 months and DNA was extracted subsequently. All 86 flies were Glossina morsitans morsitans. From these, dry RIME LAMP detected 16.3% while liquid RIME LAMP detected 11.6% as infected with trypanosomes. Ten positive samples on ITS1-PCR were sequenced and all were shown to be trypanosomes. The use of dry RIME LAMP in the field for xenomonitoring of trypanosomes in tsetse flies will greatly contribute towards control of this neglected tropical disease as it provides the cheapest, fastest and simplest way to estimate possible human infective trypanosome infection rates in the tsetse fly vectors.

Polymerase chain reaction identification of Trypanosoma brucei rhodesiense in wild tsetse flies from Nkhotakota Wildlife Reserve, Malawi.

BACKGROUND: Trypanosoma brucei rhodesiense is the causative agent of acute human African trypanosomiasis. Identification of T. b. rhodesiense in tsetse populations is essential for understanding transmission dynamics, assessng human disease risk, and monitoring spatiotemporal trends and impact of control interventions. Accurate detection and characterisation of trypanosomes in vectors relies on molecular techniques. For the first time in Malawi, a molecular technique has been used to detect trypanosomes in tsetse flies in Nkhotakota Wildlife Reserve. METHODS: A polymerase chain reaction (PCR) technique was used to identify the serum resistance associated (SRA) gene of T. b. rhodesiense in tsetse flies. Of 257 tsetse flies that were randomly caught, 42 flies were dissected for microscopic examination. The midguts of 206 flies were positive and were individually put in eppendorf tubes containing phosphate-buffered saline (PBS buffer) for DNA extraction. Internal transcribed spacer (ITS)-PCR was first used to isolate all trypanosome species from the flies. TBR PCR was then used to isolate the Trypanozoon group. T. brucei-positive samples were further evaluated by SRA PCR for the presence of the SRA gene. RESULTS: Of 257 flies caught, 185 (72%) were Glossina morsitans morsitans and 72 (28%) were Glossina pallidipes. Three were tenerals and 242 were mature live flies. Of the 242 flies dissected, 206 were positive, representing an 85.1% infection rate. From 206 infected flies, 106 (51.5%) were positive using ITS-PCR, 68 (33.0%) being mixed infections, 18 (8.7%) T. brucei, 9 (4.4%) Trypanosoma vivax, 4 (1.9%) Trypanosoma godfrey, 3 (1.5%) Trypanosoma congolense savanna, 3 (1.5%) Trypanosoma simae, and 1 (0.4%) Trypanosoma simaetsavo. When subjected to TBR PCR, 107(51.9%) were positive for T. brucei. Of the 107 T. brucei-positive samples, 5 (4.7%) were found to have the SRA gene. CONCLUSIONS: These results suggest that wild tsetse flies in Malawi are infected with human-infective trypanosomes that put communities around wildlife reserves at risk of human African trypanosomiasis outbreaks. Further studies need to be done to identify sources of blood meals for the flies and for surveillance of communities around wildlife reserves.

Glucose Metabolism Disorders, HIV and Antiretroviral Therapy among Tanzanian Adults.

INTRODUCTION: Millions of HIV-infected Africans are living longer due to long-term antiretroviral therapy (ART), yet little is known about glucose metabolism disorders in this group. We aimed to compare the prevalence of glucose metabolism disorders among HIV-infected adults on long-term ART to ART-naïve adults and HIV-negative controls, hypothesizing that the odds of glucose metabolism disorders would be 2-fold greater even after adjusting for possible confounders. METHODS: In this cross-sectional study conducted between October 2012 and April 2013, consecutive adults (>18 years) attending an HIV clinic in Tanzania were enrolled in 3 groups: 153 HIV-negative controls, 151 HIV-infected, ART-naïve, and 150 HIV-infected on ART for ≥ 2 years. The primary outcome was the prevalence of glucose metabolism disorders as determined by oral glucose tolerance testing. We compared glucose metabolism disorder prevalence between each HIV group vs. the control group by Fisher's exact test and used multivariable logistic regression to determine factors associated with glucose metabolism disorders. RESULTS: HIV-infected adults on ART had a higher prevalence of glucose metabolism disorders (49/150 (32.7%) vs.11/153 (7.2%), p<0.001) and frank diabetes mellitus (27/150 (18.0%) vs. 8/153 (5.2%), p = 0.001) than HIV-negative adults, which remained highly significant even after adjusting for age, gender, adiposity and socioeconomic status (OR = 5.72 (2.78-11.77), p<0.001). Glucose metabolism disorders were significantly associated with higher CD4+ T-cell counts. Awareness of diabetes mellitus was <25%. CONCLUSIONS: HIV-infected adults on long-term ART had 5-fold greater odds of glucose metabolism disorders than HIV-negative controls but were rarely aware of their diagnosis. Intensive glucose metabolism disorder screening and education are needed in HIV clinics in sub-Saharan Africa. Further research should determine how glucose metabolism disorders might be related to immune reconstitution.