Molecular Identification of African Trypanosome Stabilates from Livestock in Lamu County, Kenya

A study was conducted to characterize infectious trypanosomes in domestic animals within Lamu County, Kenya, using parasitological and molecular techniques. Fifteen trypanosome stabilates and 92 whole blood samples collected from parasitologically negative animals were randomly retrieved from the cryobank freezer at the Biotechnology Research Institute, (KALRO-BioRI), Kenya, and characterized. Human serum resistance associated (SRA) gene present in T. b. rhodesiense was used to differentiate T. brucei positive stabilates. Results showed that 10/15(67%) trypanosome stabilates and 13/92(14%) whole blood samples from cattle, donkeys and goats were positive using PCR. Positive T. congolense 5/23(22%) yielded a product size of 700bp using ITS1 primers. The Brucei group 7/23(30%) and T. vivax 11/23(48%) amplicons were 480bp and 250bp, respectively.  Identified trypanosome stabilates were T. b. brucei (7) T. vivax (2) and T. congolense Savannah (1). Whole blood PCR profiles revealed 13 isolates namely T. vivax (9) and T. congolense Savannah (4). Trypanosoma b. brucei, T. vivax and T. congolense Savannah were the etiological agents for AAT in donkeys. In contrast, T. vivax and T. congolense Savannah caused the disease in cattle, and T. b. brucei in goat within Lamu County. The study underscores the significance of molecular and parasitological methods during epidemiological monitoring and surveillance of disease. Keywords: Trypanosomes, livestock, PCR, Lamu County, Keny

Haematology of Experimental Trypanosoma Brucei Rhodesiense Infection in Vervet Monkeys

Haematological aberrations associated with human infective trypanosomes were investigated in the vervet monkey model of the Rhodesian sleeping sickness. Four monkeys were infected intravenously with 104 Trypanosoma brucei rhodesiense and monitored for changes in the blood profile using a haematological analyser. A chronic infection lasting between 48 and 112 days was observed. Microcytic hypochromic anaemia, which was characterized by a decline in packed cell volume (PCV), red blood cell (RBC) numbers, mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCH) developed at an early stage, and persisted throughout the infection. The mean platelet counts declined significantly from 3 x 105/\u3bcl (day 0 post infection) to 6.8 x 104/\u3bcl (day 7 post infection) and remained low in all the animals. However, the mean platelets volume rose during the course of the infection. An initial decline in total white blood cell (WBC) counts occurred between day 0 and 7 (3.1 x 106/\u3bcl) and remained low up to day 35 post infection (3.5 x 106/\u3bcl). This was followed by an increase in WBC counts, principally associated with increased lymphocyte numbers. It is concluded that microcytic hypochromic anaemia, thrombocytopaenia and an initial leucocytopaenia are the most important haematological changes associated with a chronic infection of T.b. rhodesiense infection in vervet monkeys

Pharmacology of DB844, an Orally Active aza Analogue of Pafuramidine, in a Monkey Model of Second Stage Human African Trypanosomiasis

Novel drugs to treat human African trypanosomiasis (HAT) are still urgently needed despite the recent addition of nifurtimox-eflornithine combination therapy (NECT) to WHO Model Lists of Essential Medicines against second stage HAT, where parasites have invaded the central nervous system (CNS). The pharmacology of a potential orally available lead compound, N-methoxy-6-{5-[4-(N-methoxyamidino) phenyl]-furan-2-yl}-nicotinamidine (DB844), was evaluated in a vervet monkey model of second stage HAT, following promising results in mice. DB844 was administered orally to vervet monkeys, beginning 28 days post infection (DPI) with Trypanosoma brucei rhodesiense KETRI 2537. DB844 was absorbed and converted to the active metabolite 6-[5-(4-phenylamidinophenyl)-furanyl-2-y​l]-nicotinamide(DB820), exhibiting plasma Cmax values of 430 and 190 nM for DB844 and DB820, respectively, after the 14th dose at 6 mg/kg qd. A 100-fold reduction in blood trypanosome counts was observed within 24 h of the third dose and, at the end of treatment evaluation performed four days post the last drug dose, trypanosomes were not detected in the blood or cerebrospinal fluid of any monkey. However, some animals relapsed during the 300 days of post treatment monitoring, resulting in a cure rate of 3/8 (37.5%) and 3/7 (42.9%) for the 5 mg/kg×10 days and the 6 mg/kg×14 days dose regimens respectively. These DB844 efficacy data were an improvement compared with pentamidine and pafuramidine both of which were previously shown to be non-curative in this model of CNS stage HAT. These data show that synthesis of novel diamidines with improved activity against CNS-stage HAT was possible.This investigation received financial support from the Bill and Melinda Gates Foundation through the Consortium for Parasitic Drug Development

The pathogenicity of blood stream and central nervous system forms of Trypanosoma brucei rhodesiense trypanosomes in laboratory mice: a comparative study [version 2; peer review: 2 approved]

Background: Human African trypanosomiasis (HAT) develops in two stages namely early stage when trypanosomes are found in the blood and late stage when trypanosomes are found in the central nervous system (CNS). The two environments are different with CNS environment reported as being hostile to the trypanosomes than the blood environment. The clinical symptoms manifested by the disease in the two environments are different. Information on whether blood stream are pathologically different from CNS trypanosomes is lacking. This study undertook to compare the inter-isolate pathological differences caused by bloodstream forms (BSF) and central nervous system (CNS) of five Trypanosoma brucei rhodesiense (Tbr) isolates in Swiss white mice. Methods: Donor mice infected with each of the five isolates were euthanized at 21 days post infection (DPI) for recovery of BSF trypanosomes in heart blood and CNS trypanosomes in brain supernatants. Groups of Swiss white mice (n = 10) were then infected with BSF or CNS forms of each isolate and monitored for parasitaemia, packed cell volume (PCV), body weight, survivorship, trypanosome length, gross and histopathology characteristics. Results: Amplification of SRA gene prior to trypanosome morphology and pathogenicity studies confirmed all isolates as T. b. rhodesiense. At 21 DPI, CNS trypanosomes were predominantly long slender (LS) while BSF were a mixture of short stumpy and intermediate forms. The density of BSF trypanosomes was on average 2-3 log-scales greater than that of CNS trypanosomes with isolate KETRI 2656 having the highest CNS trypanosome density. Conclusions: The pathogenicity study revealed clear differences in the virulence/pathogenicity of the five (5) isolates but no distinct and consistent differences between CNS and BSF forms of the same isolate. We also identified KETRI 2656 as a suitable isolate for acute menigo- encephalitic studies

Improved Sample Selection and Preparation Methods for Sampling Plans Used to Facilitate Rapid and Reliable Estimation of Aflatoxin in Chicken Feed

Aflatoxin B1 (AFB1), a toxic fungal metabolite associated with human and animal diseases, is a natural contaminant encountered in agricultural commodities, food and feed. Heterogeneity of AFB1 makes risk estimation a challenge. To overcome this, novel sample selection, preparation and extraction steps were designed for representative sampling of chicken feed. Accuracy, precision, limits of detection and quantification, linearity, robustness and ruggedness were used as performance criteria to validate this modification and Horwitz function for evaluating precision. A modified sampling protocol that ensured representativeness is documented, including sample selection, sampling tools, random procedures, minimum size of field-collected aggregate samples (primary sampling), procedures for mass reduction to 2 kg laboratory (secondary sampling), 25 g test portion (tertiary sampling) and 1.3 g analytical samples (quaternary sampling). The improved coning and quartering procedure described herein (for secondary and tertiary sampling) has acceptable precision, with a Horwitz ratio (HorRat = 0.3) suitable for splitting of 25 g feed aliquots from laboratory samples (tertiary sampling). The water slurring innovation (quaternary sampling) increased aflatoxin extraction efficiency to 95.1% through reduction of both bias (-4.95) and variability of recovery (1.2-1.4) and improved both intra-laboratory precision (HorRat = 1.2-1.5) and within-laboratory reproducibility (HorRat = 0.9-1.3). Optimal extraction conditions are documented. The improved procedure showed satisfactory performance, good field applicability and reduced sample analysis turnaround time

Safety, pharmacokinetic, and efficacy studies of oral DB868 in a first stage vervet monkey model of human African trypanosomiasis

There are no oral drugs for human African trypanosomiasis (HAT, sleeping sickness). A successful oral drug would have the potential to reduce or eliminate the need for patient hospitalization, thus reducing healthcare costs of HAT. The development of oral medications is a key objective of the Consortium for Parasitic Drug Development (CPDD). In this study, we investigated the safety, pharmacokinetics, and efficacy of a new orally administered CPDD diamidine prodrug, 2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868; CPD-007-10), in the vervet monkey model of first stage HAT. DB868 was well tolerated at a dose up to 30 mg/kg/day for 10 days, a cumulative dose of 300 mg/kg. Mean plasma levels of biomarkers indicative of liver injury (alanine aminotransferase, aspartate aminotransferase) were not significantly altered by drug administration. In addition, no kidney-mediated alterations in creatinine and urea concentrations were detected. Pharmacokinetic analysis of plasma confirmed that DB868 was orally available and was converted to the active compound DB829 in both uninfected and infected monkeys. Treatment of infected monkeys with DB868 began 7 days post-infection. In the infected monkeys, DB829 attained a median Cmax (dosing regimen) that was 12-fold (3 mg/kg/day for 7 days), 15-fold (10 mg/kg/day for 7 days), and 31-fold (20 mg/kg/day for 5 days) greater than the IC50 (14 nmol/L) against T. b. rhodesiense STIB900. DB868 cured all infected monkeys, even at the lowest dose tested. In conclusion, oral DB868 cured monkeys with first stage HAT at a cumulative dose 14-fold lower than the maximum tolerated dose and should be considered a lead preclinical candidate in efforts to develop a safe, short course (5-7 days), oral regimen for first stage HAT

Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model.

We assessed the virulence and anti-trypanosomal drug sensitivity patterns of Trypanosoma brucei rhodesiense (Tbr) isolates in the Kenya Agricultural and Livestock Research Organization-Biotechnology Research Institute (KALRO-BioRI) cryobank. Specifically, the study focused on Tbr clones originally isolated from the western Kenya/eastern Uganda focus of human African Trypanosomiasis (HAT). Twelve (12) Tbr clones were assessed for virulence using groups(n = 10) of Swiss White Mice monitored for 60 days post infection (dpi). Based on survival time, four classes of virulence were identified: (a) very-acute: 0-15, (b) acute: 16-30, (c) sub-acute: 31-45 and (d) chronic: 46-60 dpi. Other virulence biomarkers identified included: pre-patent period (pp), parasitaemia progression, packed cell volume (PCV) and body weight changes. The test Tbr clones together with KALRO-BioRi reference drug-resistant and drug sensitive isolates were then tested for sensitivity to melarsoprol (mel B), pentamidine, diminazene aceturate and suramin, using mice groups (n = 5) treated with single doses of each drug at 24 hours post infection. Our results showed that the clones were distributed among four classes of virulence as follows: 3/12 (very-acute), 3/12 (acute), 2/12 (sub-acute) and 4/12 (chronic) isolates. Differences in survivorship, parasitaemia progression and PCV were significant (P<0.001) and correlated. The isolate considered to be drug resistant at KALRO-BioRI, KETRI 2538, was confirmed to be resistant to melarsoprol, pentamidine and diminazene aceturate but it was not resistant to suramin. A cure rate of at least 80% was achieved for all test isolates with melarsoprol (1mg/Kg and 20 mg/kg), pentamidine (5 and 20 mg/kg), diminazene aceturate (5 mg/kg) and suramin (5 mg/kg) indicating that the isolates were not resistant to any of the drugs despite the differences in virulence. This study provides evidence of variations in virulence of Tbr clones from a single HAT focus and confirms that this variations is not a significant determinant of isolate sensitivity to anti-trypanosomal drugs

Spatial–Temporal Variations in Parasitological Prevalence and Host-Related Risk Factors of Camel Trypanosomiasis and Its Vectors in North Eastern Kenya: A Repeated Cross-Sectional Study

Camel trypanosomiasis (Surra) is endemic in the Horn of Africa. Understanding the spatiotemporal variations in Surra prevalence, vector dynamics, and host-related risk factors is important in developing effective control strategies. A repeated cross-sectional study was conducted to determine the Surra parasitological prevalence, livestock reservoirs, vector density/diversity, and host-related risk factors in Kenya. Random samples of 847, 1079, and 824 camels were screened at the start of the dry season, peak dry season, and during the rainy season, respectively. Blood samples were examined using the dark ground/phase contrast buffy-coat technique, and Trypanosoma species were identified based on their movement and morphology in wet and stained thin smears. Reservoir status for Trypanosoma evansi was assessed in 406 cattle and 372 goats. A rainy and dry seasons entomological surveys were conducted to determine the Surra vector abundance/diversity and spatiotemporal density changes. Surra prevalence was 7.1%, 3.4%, and 4.1% at the start of the dry season, peak dry season, and rainy season, respectively. Camel co-infections by Trypanozoon (T. evansi or Trypanosoma brucei brucei) and Trypanosoma vivax were recorded. Spatial variations in Surra prevalence were recorded at the beginning of dry (X7,N=8462=110.9, p≤0.001), peak dry (X7,N=10792=42.2, p≤0.001), and rainy (X7,N=8242=29.1, p≤0.001) seasons. The screened cattle and goats tested negative for Trypanozoon (T. evansi or T. b. brucei), while two cattle tested positive for Trypanosoma congolense. Biting fly catches were composed of a single species from Tabanus, Atylotus, Philoliche, Chrysops, and Stomoxys genera. The total catches for Philoliche, Chrysops, and Stomoxys were higher in the rainy than dry season consistent with the prevalence results. Surra remains an important camel disease in the region with its prevalence varying in space and time. Camel co-infections by Trypanozoon (T. evansi or T. b. brucei) and T. vivax necessitate proper diagnosis of suspected cases and targeted therapy