BACKGROUND: Human African trypanosomiasis (HAT) is a neglected tropical disease caused by Trypanosoma brucei gambiense transmitted by tsetse flies in sub-Saharan West Africa. In southern Chad the most active and persistent focus is the Mandoul focus, with 98% of the reported human cases, and where African animal trypanosomosis (AAT) is also present. Recently, a control project to eliminate tsetse flies (Glossina fuscipes fuscipes) in this focus using the sterile insect technique (SIT) was initiated. However, the release of large numbers of sterile males of G. f. fuscipes might result in a potential temporary increase in transmission of trypanosomes since male tsetse flies are also able to transmit the parasite. The objective of this work was therefore to experimentally assess the vector competence of sterile males treated with isometamidium for Trypanosoma brucei brucei.
METHODS: An experimental infection was set up in the laboratory, mimicking field conditions: the same tsetse species that is present in Mandoul was used. A T. b. brucei strain close to T. b. gambiense was used, and the ability of the sterile male tsetse flies fed on blood with and without a trypanocide to acquire and transmit trypanosomes was measured.
RESULTS: Only 2% of the experimentally infected flies developed an immature infection (midgut) while none of the flies developed a metacyclic infection of T. b. brucei in the salivary glands. We did not observe any effect of the trypanocide used (isometamidium chloride at 100 mg/l) on the development of infection in the flies.
CONCLUSIONS: Our results indicate that sterile males of the tested strain of G. f. fuscipes were unable to cyclically transmit T. b. brucei and might even be refractory to the infection. The data of the research indicate that the risk of cyclical transmission of T. brucei by sterile male G. f. fuscipes of the strain colonized at IAEA for almost 40 years appears to be small.

OBJECTIVE: African animal trypanosomiasis (AAT) is a disease affecting livestock in sub-Saharan Africa. The use of trypanocidal agents is common practice to control AAT. This study aimed to identify drug-resistant Trypanosoma congolense in Lambwe, Kenya, and assess if molecular test backed with mice tests is reliable in detecting drug sensitivity.
METHODS: Blood samples were collected from cattle, in Lambwe, subjected to buffy coat extraction and Trypanosoma spp. detected under a microscope. Field and archived isolates were subjected to molecular characterization. Species-specific T. congolense and TcoAde2 genes were amplified using PCR to detect polymorphisms. Phylogenetic analysis were performed. Four T. congolense isolates were evaluated individually in 24 test mice per isolate. Test mice were then grouped (n=6) per treatement with diminazene, homidium, isometamidium, and controls. Mice were subsequently assessed for packed cell volume (PCV) and relapses using microscopy.
RESULTS: Of 454 samples, microscopy detected 11 T. congolense spp, eight had TcoAde2 gene, six showed polymorphisms in molecular assay. Phylogenetic analysis grouped isolates into five. Two archived isolates were homidium resistant, one was also diminazene resistant in mice. Two additional isolates were sensitive to all the drugs. Interestingly, one sensitive isolate lacked polymorphisms, while the second lacked TcoAde2, indicating the gene is not involved in drug sensitivity. Decline in PCV was pronounced in relapsed isolates.
CONCLUSIONS: T. congolense associated with homidium and diminazene resistance exist in Lambwe. The impact can be their spread and AAT increase. Polymorphisms are present in Lambwe strains. TcoAde2 is unlikely involved in drug sensitivity. Molecular combined with mice tests is reliable drug sensitivity test and can be applied to other genes. Decline in PCV in infected-treated host could suggest drug resistance.

OBJECTIVE: The present study reports a very rare case of Trypanosoma theileri like infection in the peritoneal fluid of a 6-year-old female Holstein Friesian (HF) cattle brought to Veterinary Clinical Complex, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar. The aim of the clinical study was to report an unusual case of T. theileri-like infection associated with peritonitis and its therapeutic management.
METHODS: For confirmatory diagnosis, peritoneal fluid and blood sample was collected from animal for haemato-biochemical examination, buffy coat examination and detection of Trypanosoma antigen by monoclonal antibody-based latex agglutination test.
RESULTS: Animal had history of anorexia, high fever, oedematous swelling on the abdominal area and decrease in milk yield since 1 week. Clinical examination revealed high fever, congested conjunctival mucus membrane and oedematous inflammatory swelling on the abdominal region. Haematological examination on Day 1 revealed relative neutrophilia and eosinophilia, whereas other parameters measured were on the lower side of normal range. Thin blood smear examination did not reveal any parasite on first attempt. However, after confirmation of Trypanosoma species in peritoneal fluid collected by FNAC, buffy coat examination of blood sample was also found positive for Trypanosoma species. On Day 2, haematological examination revealed neutrophilic leucocytosis, eosinophilia and anaemia. Blood sample was also found serologically positive for Trypanosoma antigen by monoclonal antibody based latex agglutination test. Biochemical examination revealed increased blood urea nitrogen (BUN), gamma glutamyl transferase (GGT) levels, hyperglycemia, hyperprotienemia and decreased levels of sodium and chloride ions. Animal was primarily treated with isometamidium chloride (Nyzom®) @ 0.5 mg/kg body weight (IM, single dose) along with supportive therapy.
CONCLUSIONS: Animal showed clinical recovery after 10 days of treatment. Post-recovery, blood sample of the animal was re-examined for parasite and other blood parameters and it was found negative for presence of Trypanosoma infection along with restoration of haematological values. Hence, the present study concludes the confirmatory diagnosis and therapeutic use of isometamidium hydrochloride along with anti-inflammatory drugs to Trypanosoma infection.

African animal trypanosomosis is an important wasting and endemic protozoan disease causing morbidities and mortalities in animals in the sub-Saharan Africa. Currently, chemotherapy is the widely used method of African animal trypanosomosis control, especially in dogs in the sub-Saharan Africa. However, their efficacy is threatened by the emergence of drug-resistant trypanosomes owing to their extensive use and misuse over several decades amongst other factors. Thus, this study focused on the trypanocidal sensitivity and characterization of Trypanosoma species isolated from dogs in Enugu North Senatorial Zone (ENSZ), Southeastern Nigeria. Trypanosoma brucei (n = 44) and T. congolense (n = 4) isolated from naturally infected dogs in ENSZ, Southeastern Nigeria, between January and August 2016 were subjected to single dose test to assess their sensitivity to diminazene aceturate (DA) and isometamidium chloride (ISM). Subsequently, DA and multidrug-resistant isolates were further subjected to DA multi-dose test and CD50 was determined and was used to characterize the drug-resistant trypanosomes. Clones were derived from a randomly selected multidrug-resistant isolate and their sensitivity also assessed. 100% and 83.3% of T. congolense and T. brucei respectively were resistant to the trypanocides. Amongst the drug-resistant isolates, 50%, 16.7%, and 33.3% were resistant to DA, ISM, and both trypanocides respectively with CD50 ranging between 11 and 32.34 mg/kg. Drug-resistant trypanosomes were characterized into highly resistant (CD50 = 11-24.99 mg/kg) and very highly resistant (CD50 =  > 25 mg/kg) trypanosome isolates. Clones also expressed high levels of resistance to both DA and ISM with CD50 values between 35.58 and 38.85 mg/kg. Trypanocidal resistance was, thus, confirmed and appears to be widespread in dogs in ENSZ, Southeastern Nigeria. The adoption of an integrated trypanosomosis control strategy in ENSZ is most desirous.

Trypanosoma congolense is a principal agent causing livestock trypanosomiasis in Africa, costing developing economies billions of dollars and undermining food security. Only the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induced stable diminazene resistance in T. congolense strain IL3000 in vitro. There was no cross-resistance with the phenanthridine drugs, melaminophenyl arsenicals, oxaborole trypanocides, or with diamidine trypanocides, except the close analogs DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [3 H]-diminazene was slow with low affinity and partly but reciprocally inhibited by folate and by competing diamidines. Expression of T. congolense folate transporters in diminazene-resistant Trypanosoma brucei brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [3 H]-diminazene transport studies, whole-genome sequencing, and RNA-seq found no major changes in diminazene uptake, folate transporter sequence, or expression. Instead, all resistant clones displayed a moderate reduction in the mitochondrial membrane potential Ψm. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters; while resistance is associated with a reduction in Ψm it is unclear whether this is the primary cause of the resistance.

The four components present in the trypanocidal treatment Samorin, the commercially available formulation of isometamidium, were separated and purified by column chromatography. These compounds as well as the Samorin mixture and the other phenanthridine trypanocide, homidium, were tested on Trypanosoma congolense and wild type, diamidine- and isometamidium-resistant Trypanosoma brucei brucei strains using an Alamar blue drug sensitivity assay. EC50 values obtained suggest that M&B4180A (2) was the most active of the components, followed by M&B38897 (1) in all the strains tested, whereas M&B4596 (4) was inactive. Samorin was found to be significantly more active than any of the individual components alone, against T. congolense and all three T. b, brucei strains. Samorin and all its active constituents displayed reduced activity against the previously characterised isometamidium-resistant strain ISMR1.

Trypanosoma congolense is a protozoan parasite that is transmitted by tsetse flies, causing African Animal Trypanosomiasis, also known as Nagana, in sub-Saharan Africa. Nagana is a fatal disease of livestock that causes severe economic losses. Two drugs are available, diminazene and isometamidium, yet successful treatment is jeopardized by drug resistant T. congolense. Isothermal microcalorimetry is a highly sensitive tool that can be used to study growth of the extracellular T. congolense parasites or to study parasite growth inhibition after the addition of antitrypanosomal drugs. Time of drug action and time to kill can be quantified in a simple way by real time heat flow measurements. We established a robust protocol for the microcalorimetric studies of T. congolense and developed mathematical computations in R to calculate different parameters related to growth and the kinetics of drug action. We demonstrate the feasibility and benefit of the method exemplary with the two standard drugs, diminazene aceturate and isometamidium chloride. The method and the mathematical approach can be translated to study other pathogenic or non-pathogenic cells if they are metabolically active and grow under axenic conditions.

BACKGROUND: Trypanocidal drugs have been used to control African animal trypanosomosis for several decades. In Ethiopia, these drugs are available from both authorized (legal) and unauthorized (illegal) sources but documentation on utilization practices and quality of circulating products is scanty. This study looked at the practices of trypanocidal drug utilization by farmers and the integrity of active ingredient in trypanocides sold in Gurage zone, south western Ethiopia. The surveys were based on a structured questionnaire and drug quality determination of commonly used brands originating from European and Asian companies and sold at both authorized and unauthorized markets. One hundred farmers were interviewed and 50 drug samples were collected in 2013 (Diminazene aceturate = 33 and Isometamidium chloride = 17; 25 from authorized and 25 from unauthorized sources). Samples were tested at the OIE-certified Veterinary Drug Control Laboratory (LACOMEV) in Dakar, Senegal, by using galenic standards and high performance liquid chromatography.
RESULTS: Trypanosomosis was found to be a major threat according to all interviewed livestock keepers in the study area. Diminazene aceturate and isometamidium chloride were preferred by 79% and 21% of the respondents respectively, and 85% of them indicated that an animal receives more than six treatments per year. About 60% of these treatments were reported to be administered by untrained farmers. Trypanocidal drug sources included both unauthorized outlets (56%) and authorized government and private sources (44%). A wide availability and usage of substandard quality drugs was revealed. Twenty eight percent of trypanocidal drugs tested failed to comply with quality requirements. There was no significant difference in the frequency of non-compliance between diminazene-based and isometamidium chloride products (P = 0.87) irrespective of the marketing channel (official and unofficial). However, higher rates of non-compliant trypanocides were detected for drugs originating from Asia than from Europe (P = 0.029).
CONCLUSIONS: The findings revealed the presence of risk factors for the development of drug resistance, i.e. wide distribution of poor quality drugs as well as substandard administration practices. Therefore, it is strongly recommended to enforce regulatory measures for quality control of veterinary drugs, to expand and strengthen veterinary services and to undertake trypanocidal drug efficacy studies of wider coverage.

Isometamidium hydrochloride (ISMM) is an effective drug for the treatment of trypanosomosis, but it causes local and systemic toxicity. Isometamidium hydrochloride has limited therapeutic index and exhibit considerable variation in their prophylactic activities. We developed a trypanocidal nanoformulation using ISMM and polymers sodium alginate/gum acacia to enhance the efficacy of the drug at lower doses, while minimizing undesirable side effects. It was characterized by transmission electron microscopy and infrared spectroscopy for evaluation of size, morphology, functional groups, etc. In vitro cytotoxicity studies were performed by metabolic resazurin assay at different concentrations of isometamidium-loaded alginate/gum acacia nanoparticles using equine peripheral blood mononuclear cells. Hemolytic assay revealed significantly less toxicity compared to the conventional drug. The results demonstrate that the developed drug delivery module can be evaluated in suitable animal models to evaluate its potency.

Trypanocidal drugs remain the most accessible and thus commonly used means of controlling tsetse transmitted animal African trypanosomosis. In Togo, trypanocides are sold on official as well as unofficial markets, but the quality of these trypanocides is undocumented so a drug quality assessment study was conducted from May 2013 to June 2014. Trypanocides supplied by European, Indian and Chinese pharmaceutical companies and sold on official and unofficial markets in Togo were purchased. In total fifty-two trypanocides were obtained, 24 of these samples from official markets and 28 from unofficial markets made up of a total of 36 diminazene diaceturate and 16 isometamidium chloride hydrochloride samples. The samples were analysed in the reference laboratory of the OIE (World Organisation for Animal Health), Laboratory for the Control of Veterinary Medicines (LACOMEV) in Dakar which uses galenic testing and high performance liquid chromatography (HPLC) testing as standard reference analysis methods. The results revealed a high proportion of trypanocides of sub-standard quality on the Togolese market: 40% were non-compliant to these quality reference standards. All of the HPLC non-compliant samples contained lower amounts of active ingredient compared to the concentration specified on the packaging. Non-compliance was higher in samples from the unofficial (53.57%) than from the official markets (25%; p=0.04).The main drug manufacturers, mostly of French origin in the study area, supply quality drugs through the official legal distribution circuit. Products of other origins mostly found on illegal markets present a significantly lower quality.