Quality, supply chain, and use of Trypanocidal drugs among camel keepers in north-eastern Kenya: A cross-sectional study.

The non-cyclic trypanosomiasis (surra), caused by Trypanosoma evansi, and mechanically transmitted by biting flies, hinders camel productivity in Kenya. Trypanocides are the most commonly used drugs to control surra. However, emergence of drug resistance by the parasites is a major limitation to control efforts. There is limited information on the quality of trypanocides, the supply chain and drug-use practices among camel keepers potentially contributing to development of drug resistance in Kenya. We sought to fill this gap by conducting a cross-sectional study among camel keepers in Isiolo and Marsabit counties, Kenya. We mapped the trypanocide drugs supply chain through quantitative and qualitative surveys. We administered a semi-structured questionnaire to camel keepers to generate data on trypanocides-use practices, including the types, sources, person who administers treatment, reconstitution, dosage, route and frequency of administration, among others. Additionally, we tested the quality of trypanocidal drugs retailed in the region. We mapped a total of 55 and 49 agro-veterinary outlets and general (ordinary) shops retailing veterinary drugs in the two counties, respectively. These comprised of 29 and 26 agro-veterinary outlets, as well as 24 and 25 general shops in Isiolo and Marsabit counties, respectively. Overall, the respondents experienced 283 surra cases in the three-month recall period, which were treated with trypanocides. The majority of these cases were diagnosed by camel owners (71.7%) and herders (24.1%). A significant proportion of the cases were treated by camel owners (54.8%), herders (35.3%), the owner's son (3.2%) and veterinary personnel (1.1%) (χ2 = 24.99, p = 0.000). Most of the households sourced the drugs from agro-veterinary outlets (59.0%), followed by general shops (19.8%), veterinary personnel (2.1%), and open-air markets (0.4%) (χ2 = 319.24, p = 0.000). Quinapyramine was the most (56.9%) predominantly used trypanocide in treatment of surra, followed by homidium (19.8%), isometamidium (15.9%), diminazene aceturate (6.7%), and ethidium (0.7%) (χ2 = 340.75, p < 0.000). Only a meager proportion of respondents (15.2%) used the drugs correctly as instructed by the manufacturers. We recorded an association between correct drug usage, with the person who administers the treatment (χ2 = 17.7, p = 0.003), and the type of trypanocide used (χ2 = 19.4, p < 0.001). All the drug samples tested had correct concentrations of active ingredient (100.0%), and therefore of good quality. We have demonstrated that whereas the trypanocides retailed in the region by authorized vendors are of good quality, there is widespread incorrect handling and use of the drugs by unqualified individuals, which may contribute to treatment failure and emergence of trypanocide resistance.

Isometamidium chloride alters redox status, down-regulates p53 and PARP1 genes while modulating at proteomic level in Drosophila melanogaster.

As trypanocide, several side effects have been reported in the use of Isometamidium chloride. This study was therefore, designed to evaluate its ability to induce oxidative stress and DNA damage using D. melanogaster as a model organism. The LC50 of the drug was determined by exposing the flies (1-3 days old of both genders) to six different concentrations (1 mg, 10 mg, 20 mg, 40 mg, 50 mg and 100 mg per 10 g of diet) of the drug for a period of seven days. The effect of the drug on survival (28 days), climbing behavior, redox status, oxidative DNA lesion, expression of p53 and PARP1 (Poly-ADP-Ribose Polymerase-1) genes after five days exposure of flies to 4.49 mg, 8.97 mg, 17.94 mg and 35.88 mg per 10 g diet was evaluated. The interaction of the drug in silico with p53 and PARP1 proteins was also evaluated. The result showed the LC50 of isometamidium chloride to be 35.88 mg per 10 g diet for seven days. Twenty-eight (28) days of exposure to isometamidium chloride showed a decreased percentage survival in a time and concentration-dependent manner. Isometamidium chloride significantly (p < 0.05) reduced climbing ability, total thiol level, Glutathione-S-transferase, and Catalase activity. The level of H2O2 was significantly (p < 0.05) increased. The result also showed significant (p < 0.05) reduction in the relative mRNA levels of p53 and PARP1 genes. The in silico molecular docking of isometamidium with p53 and PARP1 proteins showed high binding energy of -9.4 Kcal/mol and -9.2 Kcal/mol respectively. The results suggest that isometamidium chloride could be cytotoxic and a potential inhibitor of p53 and PARP1 proteins.

Vector competence of sterile male Glossina fuscipes fuscipes for Trypanosoma brucei brucei: implications for the implementation of the sterile insect technique in a sleeping sickness focus in Chad.

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.

Nanoformulation of a Trypanocidal Drug Isometamidium Chloride Ameliorates the Apurinic-Apyrimidinic DNA Sites/Genotoxic Effects in Horse Blood Cells.

Isometamidium chloride (ISM) is a trypanocide for the prophylactic and therapeutic use against vector-borne animal trypanosomosis (mainly Surra caused by Trypanosoma evansi) and African animal trypanosomosis caused by T. congolense/T. vivax/T. brucei). ISM was found to be an efficient trypanocide for therapeutic/prophylactic use against trypanosomosis; however, it produces some local and systemic detrimental effects in animals. We synthesized isometamidium chloride-loaded alginate gum acacia nanoformulation (ISM SANPS) to lessen the detrimental side effects of isometamidium chloride (ISM) while treating trypanosomal diseases. We intended to determine the cytocompatibility/toxicity, and DNA deterioration/chromosomal structural or number changes (genotoxicity) of ISM SANPs using mammalian cells in a concentration-dependent manner. Apurinic/apyrimidinic (AP) sites are one of the major types of DNA lesions formed during base excision and repair of oxidized, deaminated, or alkylated bases. The intensity of the cellular AP site is an excellent marker of the deterioration of DNA quality. We thought it pertinent to quantify the AP sites in ISM SANPs-treated cells. Our investigations established a dose-dependent cyto-compatibility or toxicity and DNA impairment (genotoxicity) in ISM SANPs-treated horse peripheral blood mononuclear cells. ISM SANPs were biocompatible at various concentrations tested on the mammalian cells.

Trypanosoma Congolense Resistant to Trypanocidal Drugs Homidium and Diminazene and their Molecular Characterization in Lambwe, Kenya.

PURPOSE: 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. CONCLUSION: 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.

Clinical Diagnosis and Therapeutic Management of Trypanosoma theileri Infection Associated with Peritonitis in Crossbred Cow: A Rare Case Report.

PURPOSE: 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. CONCLUSION: 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.

In vivo analysis of trypanocidal drug resistance in sahelian goats infected by Trypanosoma vivax strains collected in northern Togo.

Trypanosoma (T.) vivax is one of the animal trypanosomes species causing calf mortality and economic losses in Togo. Despite its importance as the most widely distributed trypanosome species, T. vivax has received little attention because it is difficult to cultivate most field isolates in rodents. No molecular diagnostic tools for the identification of drug-resistant in T. vivax are currently available. Herein, four field isolates of T. vivax from Togo were cryopreserved and assessed for susceptibility to diminazene aceturate (DA) and isometamidium chloride (ISM) in goats. For field isolate preparation, 1 ml of blood from an infected goat was diluted in 111 µl of phosphate-buffered-saline and stored in liquid nitrogen. The in vivo experiment drug test was performed using twenty Sahelian goats with six-month of age and weighing 14.5 ± 1.6 kg. These experimental goats were purchased from a tsetse free-area Dori, a Sahelian region of Burkina Faso. The cryopreserved T. vivax isolates with unknowns, DA, and ISM sensitivity was inoculated to five goats and one goat was used as control. Each animal was inoculated by intravenously route 1 × 105 trypanosomes from the donor goat. Relapses were earlier in the first phase of treatment (14.85 ± 1.08 days) compared with the second phase (20 ± 3.39 days). The overall mean PCV of the control group decreased from 32% to 17% at day-60 (P-value < 0.001). Three isolates were phenotypically resistant to 0.5 mg per kg body weight (BW) ISM and one for 3.5 mg per kg BW of DA. There were no relapses with the 7 mg per kg BW dose DA. This study shows the resistance of T. vivax to two main trypanocidal drugs in different villages of Mango. The results suggest the extension of surveillance strategies to remote villages in Togo and will guide the veterinarian or herder in choosing a mass treatment strategy. Further studies will be needed to better understand the molecular basis of the observed resistance.

Drug-resistant trypanosome isolates populations in dogs in Enugu North Senatorial Zone, Southeastern Nigeria.

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.

Development of cationic Isometamidium chloride loaded long-acting lipid nanoformulation: optimization, cellular uptake, pharmacokinetics, biodistribution, and immunohistochemical evaluation.

The aim of the present work involved the development and evaluation of long-acting Isometamidium chloride (ISMM)-Docusate sodium (DS) complex loaded lipid nanoparticles (LA ISMM-DS LNP). The development involved screening various anionic complexing agents, including DS, dextran sulphate, and sodium alginate. Anionic DS was selected to synthesize hydrophobic ionic complex (ISMM-DS HIC), which was loaded into lipid nanoparticles (LA ISMM-DS LNP) by in situ complexation followed by the solvent evaporation method. 35-5-folds increase in the drug loading of hydrophilic cationic ISMM within nanoparticles was observed due to ISMM-DS HIC. The LA ISMM-DS LNP were non-hemolytic (0-2.52%), cytocompatible (80.6-47.5% cell viability), and enhanced THP-1 cellular uptake (2.3-folds higher) compared with free ISMM. The LA ISMM-DS LNP engender protracted in vivo plasma drug concentration for seven days with enhanced AUC0-ꝏ, MRT0-ꝏ, and t1/2, along with reduced Cl compared with free ISMM. Interestingly, the amount of ISMM was 2.9-, 4.2- and 2.0-folds higher in target reticuloendothelial (RES) organs like liver (Kupffer cells), spleen (spleenotropic macrophages and 15% T-lymphocytes), and lymph nodes (75% T-lymphocytes), respectively in LA ISMM-DS LNP group compared with free ISMM. Furthermore, LA ISMM-DS LNP caused higher peripheral blood mononuclear cells (PBMC) infiltration with diminished toxicity and inflammation. Therefore, the in vitro and in vivo studies predicted enhanced safety and efficacy of LA ISMM-DS LNP compared with free ISMM. To conclude, successfully developed LA ISMM-DS LNP would elicit a tremendous clinical potential for treatment and prevention against trypanosomiasis.

Diminazene resistance in Trypanosoma congolense is not caused by reduced transport capacity but associated with reduced mitochondrial membrane potential.

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.

Efficacy of diminazene diaceturate and isometamidium chloride hydrochloride for the treatment of Trypanosoma evansi in mice model.

Diminazene diaceturate (DIM) and isometamidium chloride hydrochloride (ISMM) have been widely used for the treatment of animal trypanosomosis. We evaluated the efficacy of standard doses of DIM and ISMM followed by their double doses for the treatment of Trypanosoma evansi in experimentally infected mice. A T. evansi strain obtained from a naturally infected camel in Afar was used. 25 swiss white mice randomly divided in to five groups were inoculated with 0.2 mL of blood containing 103 trypanosomes. At the peak of parasitemia (≈ 2 weeks post infection), groups A and B were treated with the standard dose (3.5 mg/kg body weight [BWT]) of DIM; groups C and D were treated with the standard dose (0.5 mg/kg BWT) of ISMM; and group E served as infected control. In the DIM standard dose groups, relapses and peak parasitemia were observed 20- and 25-days post treatment respectively. Similarly, relapses and peak parasitemia were observed 21- and 27-days post treatment in the ISMM standard dose groups. All mice in the control group died within two weeks post infection. Following relapses, mice were treated with the double doses of DIM (7 mg/kg BWT) or ISMM (1 mg/kg BWT). Parasitemia was not detected for 3 months following the double dose treatments. Following dexamethasone administration for 7 days, all but one mouse in the DIM group remained negative for another month. In general, although the T. evansi strain was resistant to the standard doses of DIM and ISMM their double doses completely cleared the infection.

Occurrence of Trypanosoma spp. and piroplasm infections of horses at Obollo-Afor southeastern Nigeria and resistance profiles of trypanosomes to isometamidium and diminazene salts.

Epidemiological data on trypanosomosis and piroplasmosis of horses are lacking in southeastern Nigeria. The prevalence of trypanosome and piroplasm infections in horses and resistance profile of isolated trypanosomes to diminazene and isometamidium salts were investigated. For the cross-sectional study of horses billed for slaughter, 304 horses were randomly sampled. Approximately 2 ml of blood was collected into anticoagulant-treated bottles for haematocrit (HCT) determination, direct microscopic examinations, and rat inoculation. Gender, body condition scores (BCS), age groups, and body weights of sampled horses were noted. Two isolates of Trypanosoma brucei recovered from the cross-sectional study were profiled for resistance to isometamidium hydrochloride and diminazene diaceturate in 36 BALB/c mice. Standardized protocols were used (Eisler et al., Veterinary Parasitology 97:171-182, 2001). 19.1% of horses (95% confidence interval 14.7-23.5%) were positive for haemoparasite infections including Theileria equi (16.1%) and Babesia caballi (3.9%). Only two (0.66%) Trypanosoma brucei infections were seen, being from active cases. Associations between age or gender, and presence of haemoparasites were only random. Haemoparasite-infected horses had significantly (p < 0.05) lower mean HCT and body weights and poorer BCS. From resistance profiling, for each isolate, all mice in control groups were parasitaemic by day 6 post-inoculation, while mice in test groups remained aparasitaemic over 60-day observation period. The study showed the endemicity and weights of Trypanososma spp. and piroplasm infections and among horses within the area. Furthermore, circulating strains of Trypanosoma brucei in the area are still susceptible to isometamidium and diminazene salts in mice. The pharmacoepidemiological significances of these findings were discussed.

Comparison of therapeutic efficacy of different drugs against Trypanosoma vivax on experimentally infected cattle.

In this study, we evaluated the therapeutic efficacy of diminazene diaceturate at a dose of 7 mg/kg (DA), imidocarb dipropionate at 4.8 mg/kg (IMD), isometamidium chloride at 0.5 and 1.0 mg/kg (ISM 0.5 and ISM 1.0) and combinations applied through different methods to treat Trypanosoma vivax in experimentally infected calves. Thirty male Girolando calves were kept indoors and infected intravenously with T. vivax trypomastigotes (approximately 1 × 106). On D-1, the calves were randomized based on the quantity of infecting parasites per animal, yielding six groups of five animals each: G1: positive control group without treatment; G2 animals treated with DA on Day 0 intramuscularly (IM); G3 animals treated with IMD on Day 0 and D + 14 subcutaneously; G4 animals treated with ISM 0.5 on Day 0 IM; G5 animals treated with ISM 1.0 on Day 0 IM; G6 animals received DA on Day 0 and ISM 1.0 on D + 14, both IM. Throughout 180 days, blood samples were collected for the evaluation of T. vivax using the Woo, Brener and PCR methods. The results indicated that the treatment protocols with DA and/or ISM 0.5 and ISM 1.0 had high efficacy (100 %) against T. vivax. Interestingly, cattle that received ISM remained free of parasites until D + 180. In contrast, animals treated with IMD had relapsed T. vivax detected on the 10th and 14th days post-treatment (DPT). Cattle that received ISM 1.0 did not exhibit relapsed T. vivax in the blood, even after reinfection performed on the 50th DPT. However, treatment with DA on Day 0 failed to prevent a new infection of T. vivax on the 50th DPT. The animals that received ISM 1.0 had a transient decrease in packed cell volume similar to that found in the control group. The reappearance of T. vivax in herds in Brazil treated with DA likely occurred due to the short half-life of the drug and not necessarily due to T. vivax resistance to DA.

Molecular identification of diminazene aceturate-resistant strains of Trypanosoma congolense in naturally infected domestic animals of Yoko in the centre region of Cameroon.

African animal trypanosomiases (AAT) remain the major constraint for livestock production, agriculture and food security in Africa. Although several control measures have been developed to fight AAT, the use of trypanocides remains the main strategy in most affected poor and rural communities. However, several studies have highlighted drug-resistant-trypanosome infections in many African countries, though this phenomenon is still not well described. This study aims to detect trypanosome species and the molecular profiles of drug-resistant-trypanosomes in naturally infected domestic animals of Yoko in the centre region of southern Cameroon. Therefore, in October 2017, 348 animals were blood sampled. The level of packed cell volume (PCV) was evaluated in each animal and trypanosome infections were investigated with the capillary tube centrifugation technique (CTC). Thereafter, DNA was extracted from blood samples and different trypanosome species were identified by PCR. The resistant/sensitive molecular profiles of trypanosomes for diminazene aceturate (DA) and isometamidium chloride (ISM) were investigated by PCR-RFLP. About 18.4% (64/348) of animals analyzed by PCR were found with trypanosome infections including Trypanosoma vivax, Trypanosoma brucei s.l. and Trypanosoma congolense forest and savannah. Trypanosoma congolense savannah was the predominant species with an infection rate of 15.2%. Between villages, significant (p˂0.0001) differences were found in the overall trypanosome infection rates. No molecular profile for ISM resistant-trypanosomes was identified. Conversely, about 88.9% (40/45) of T. congolense positive samples have shown molecular profiles of DA-resistant strains while the remaining 11.1% (5/45) showed mixed molecular profiles of resistant/sensitive strains. Results showed that the molecular profiles of DA-resistant strains of T. congolense in domestic animals of Yoko were widespread. This data needs to be confirmed by testing in vivo the drug susceptibilities of the trypanosome strains herein detected. In conclusion, appropriate future control measures are required. In addition to the intensification of vector control, ISM is advised for the treatment of animals infected by trypanosomes.

Factors associated with persistence of African animal trypanosomiasis in Lango subregion, northern Uganda.

African animal trypanosomiasis (AAT) continues to inflict heavy losses on livestock production especially cattle in terms of decreased production and productivity in Uganda. AAT is a disease complex caused by tsetse fly-transmitted Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense, Trypanosoma congolense, and Trypanosoma vivax. The disease is most important in cattle but also known to cause serious losses in pigs, camels, goats, and sheep. Several control measures including live bait technology, mass treatment of cattle with trypanocidal drugs, and deployment of tsetse traps have been used in the past 10 years, but the problem still persists in some areas. This necessitated an exploration of the factors associated with continued trypanosome infections in cattle, which are also known reservoirs for the zoonotic trypanosomiasis. A structured questionnaire was administered to 286 animal owners from 20 villages purposively selected from Lira, Kole, and Alebtong districts of Lango subregion to obtain information on the factors associated with persistence of infection. Over 50% of the respondents reported trypanosomiasis as a major challenge to their livestock. Land ownership (P = 0.029), type of livestock kept (P = 0.000), disease control strategy employed (P = 0.000), source of drugs (P = 0.046), and drug preparation (P = 0.017) were associated with persistent AAT infection. We recommend continued farmer sensitization on the threat of AAT and the available prevention and control options. The use of isometamidium chloride for prophylaxis against trypanosomiasis is highly recommended. There is also a need to foster qualified private veterinary drug supply in the region.

The individual components of commercial isometamidium do not possess stronger trypanocidal activity than the mixture, nor bypass isometamidium 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.

Isothermal microcalorimetry - A quantitative method to monitor Trypanosoma congolense growth and growth inhibition by trypanocidal drugs in real time.

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.

Aberrant use and poor quality of trypanocides: a risk for drug resistance in south western Ethiopia.

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). CONCLUSION: 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.

Genomic analysis of Isometamidium Chloride resistance in Trypanosoma congolense.

Isometamidium Chloride (ISM) is one of the principal drugs used to counteract Trypanosoma congolense infection in livestock, both as a prophylactic as well as a curative treatment. However, numerous cases of ISM resistance have been reported in different African regions, representing a significant constraint in the battle against Animal African Trypanosomiasis. In order to identify genetic signatures associated with ISM resistance in T. congolense, the sensitive strain MSOROM7 was selected for induction of ISM resistance in a murine host. Administered ISM concentrations in immune-suppressed mice were gradually increased from 0.001 mg/kg to 1 mg/kg, the maximal dose used in livestock. As a result, three independent MSOROM7 lines acquired full resistance to this concentration after five months of induction, and retained this full resistant phenotype following a six months period without drug pressure. In contrast, parasites did not acquire ISM resistance in immune-competent animals, even after more than two years under ISM pressure, suggesting that the development of full ISM resistance is strongly enhanced when the host immune response is compromised. Genomic analyses comparing the ISM resistant lines with the parental sensitive line identified shifts in read depth at heterozygous loci in genes coding for different transporters and transmembrane products, and several of these shifts were also found within natural ISM resistant isolates. These findings suggested that the transport and accumulation of ISM inside the resistant parasites may be modified, which was confirmed by flow cytometry and ex vivo ISM uptake assays that showed a decrease in the accumulation of ISM in the resistant parasites.

[Simultaneous determination of diminazene aceturate and isometamidium chloride residues in cattle tissues by high performance liquid chromatography with solid-phase extraction].

A method for the simultaneous determination of trypanocidal diminazene aceturate (DIM) and isometamidium chloride (ISM) that containing benzamidine groups in cattle tissues was developed by high performance liquid chromatography (HPLC) with solid-phase extraction (SPE). The tissue samples were extracted with different proportions of water-acetonitrile, then were cleaned up by Oasis WCX cartridges. DIM and ISM were separated by HPLC with a Spherisorb CN column (250 mm×4.6 mm, 5 µm). Acetonitrile-0.05 mol/L ammonium formate solution (pH 2.4) was used as mobile phases with gradient elution. The detection wavelength of UV was set at 380 nm. The limits of detection (LODs) and the limits of quantification (LOQs) of DIM and ISM in cattle tissues were 0.01 mg/kg and 0.025 mg/kg, respectively. The correlation coefficients (r) of DIM and ISM in cattle tissues were not less than 0.9993. The average recoveries of DIM and ISM at three spiked levels were 82.2%-97.6% with the intra-day relative standard derivations (RSDs) of 0.3%-5.2% (n=5) and inter-day RSDs of 1.3%-5.2% (n=15). The method was successfully applied to the analysis of DIM and ISM in cattle tissues. The method is rapid, sensitive and repeatable for the determination of diminazene aceturate and isometamidium chloride in cattle tissues.