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 (X (7, N = 846) 2 = 110.9, p ≤ 0.001), peak dry (X (7, N = 1079) 2 = 42.2, p ≤ 0.001), and rainy (X (7, N = 824) 2 = 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.

Coenzyme Q10 prevented Trypanosoma brucei rhodesiense-mediated breach of the blood brain barrier, inflammation and organ damage in late stage of Human African Trypanosomiasis.

During the late stage of Human African Trypanosomiasis (HAT), there is severe cytokine-driven inflammation, oxidative stress and organ damage. Controlling inflammation and oxidative damage presents unique therapeutic opportunities to improve treatment outcome. The current study sought to determine the putative impact of Coenzyme-Q10 (Co-Q10), a potent antioxidant and anti-inflammatory, on adverse inflammatory and oxidative events during Trypanosoma brucei rhodesiense (T.b.r) infection. Group one constituted the control; the second group was infected with T.b.r; the third group was orally administered with 200 mg/kg Co-Q10 for two weeks; thereafter, Co-Q10 administration continued after infection with T.b.r. Co-Q10 improved the survival rate of infected mice and prevented full blown parasite driven splenomegaly and hepatomegaly. Co-Q10 prevented characteristic T.b.r-driven breach of the blood brain barrier and improved neurological integrity among T.b.r infected mice. Co-Q10 protected from T.b.r-induced microcytic hypochromic anaemia and thrombocytopenia. T.b.r-induced oxidative stress in the vital organs was assuaged following exposure to Co-Q10. Co-Q10 blocked T.b.r-induced derangement of high density lipoprotein and triglyceride levels. Co-Q10 significantly abrogated T.b.r-driven elevation of serum TNF-α and IFN-γ levels. Moreover, T.b.r-induced kidney and liver damage was assuaged by Co-Q10 administration. Co-Q10 administration downregulated T.b.r-induced elevation of uric acid and C-reactive protein. Likewise, T.b.r infected mice receiving Co-Q10 exhibited normal brain architecture. In conclusion, treatment with Co-Q10 may be useful in protecting against T.b.r-mediated organ injury, lethal inflammation and oxidative stress commonly present in severe late stage HAT; and presents unique opportunities for an adjunct therapy for late stage HAT.

Variation of sensitivity of Trypanosoma evansi isolates from Isiolo and Marsabit counties of Kenya to locally available trypanocidal drugs.

Trypanocidal resistance is a major cause of treatment failure. This study evaluated the sensitivity of Trypanosoma evansi field isolates collected from Marsabit and Isiolo counties, Kenya. A total of 2,750 camels were screened using parasitological tests for trypanosomes. Of the screened camels, 113 tested positive from which 40 T. evansi isolates were tested using the single dose mice sensitivity test. Five treatment groups each comprising of 6 mice were inoculated intraperitoneally with 1x105 trypanosomes of each isolate and treated 24 hours later with isometamidium chloride at 1 mg/kg, homidium chloride at 1mg/kg, diminazene aceturate at 20 mg/kg and quinapyramine sulphate & chloride at 1 mg/kg. The fifth group was left untreated (positive control). The mice were monitored daily for 60 days. A survey on camel owners' practices that influence development of resistance to trypanocidal drugs was then conducted. Results indicated presence of drug resistance in all the 7 study sites that had infected camels. Seven of the isolates tested were resistant to diminazene aceturate whereas, 28, 33 and 34 were resistant to isometamidium chloride, quinapyramine sulphate & chloride and homidium chloride, respectively. Seven (17.5%) isolates of the 40 tested were sensitive to all 4 drugs, whereas, 7.5%, 10%,55% and 10% were resistant to 1,2,3 and 4 drugs, respectively. The prevalence of multiple drug resistance was 75%. Survey data indicated that camel management practices influenced the prevalence and degree of drug resistance. In conclusion, the multiple drug resistance observed in the two counties may not be an indication of total trypanocidal drug failure. Judicious treatment of confirmed trypanosomiasis cases with correct dosage would still be effective in controlling the disease since the observed resistance was at the population and not clonal level. However, integrated control of the disease and the vectors using available alternative methods is recommended to reduce drug use.

The pathogenicity of blood stream and central nervous system forms of Trypanosoma brucei rhodesiense trypanosomes in laboratory mice: a comparative study.

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.

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.

Comparative pathogenicity of Trypanosoma brucei rhodesiense strains in Swiss white mice and Mastomys natalensis rats.

We evaluated Mastomys natelensis rat as an animal model for Rhodesian sleeping sickness. Parasitaemia, clinical and pathological characteristics induced by T. b. rhodesiense isolates, KETRI 3439, 3622 and 3637 were compared in Mastomys rats and Swiss white mice. Each isolate was intra-peritonially injected in mice and rat groups (n=12) at 1×10(4) trypanosomes/0.2mL. Pre-patent period (PP) range for KETRI 3439 and KETRI 3622-groups was 3-6 days for mice and 4-5 days for rats while for KETRI 3637-infected mice and rats was 5-9 and 4-12 days, respectively. Pairwise comparison between PP of mice and rats separately infected with either isolate showed no significant difference (p>0.05). The PP's of KETRI 3637-infected mice were significantly (p>0.01) longer than those infected with KETRI 3439 or KETRI 3622, a trend also observed in rats. The second parasitaemic wave was more prominent in mice. Clinical signs included body weakness, dyspnoea, peri-orbital oedema and extreme emaciation which were more common in rats. Survival time for KETRI 3439 and 3622-infected groups was significantly (p<0.05) longer in mice than rats but similar in KETRI 3637-infected groups. Inflammatory lesions were more severe in rats than mice. All mice and KETRI 3622-infected rats had splenomegaly, organ congestion with rats additionally showing prominent lymphadenopathy. KETRI 3439-infected rats showed hemorrhagic pneumonia, enteritis with moderate splenomegaly and lymphadenopathy. KETRI 3637-infected rats had the most severe lesions characterized by prominent splenomegaly, lymphadenopathy, hepatomegaly, enlarged adrenal glands, organ congestion, generalized oedemas, gastroenteritis, pneumonia and brain congestion. KETRI 3637-infected Mastomys is a suitable model for studying pathophysiology of HAT.

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 C(max) (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.

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-yl]-nicotinamide (DB820), exhibiting plasma C(max) 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.

A competitive enzyme-linked immunosorbent assay for diminazene.

Diminazene aceturate has remained a very important therapeutic drug for trypanosomosis in cattle, sheep and goats since its introduction into the market in 1955. Despite its continued use, the methods available for its detection in body fluids are lengthy and inefficient for routine monitoring of drug levels in treated animals. A competitive enzyme linked immunosorbent assay (ELISA) has now been developed and optimized for the detection of diminazene in bovine serum. In the assay, diminazene in the test samples and that in a newly developed diminazene-horseradish peroxidase conjugate compete for antibodies to diminazene raised in rabbits and immobilized on a microtitre plate. Tetramethylbenzidine-hydrogen peroxide (TMB/H(2)O(2)) is used as chromogen-substrate system. The assay has a detection limit of 0.8 ng/ml of serum with a high specificity for diminazene. Cross-reactivity with either homidium bromide and quinapyramine sulphate/chloride of 0.0004% is negligible while that with isometamidium chloride is 0.71%. The assay was able to detect diminazene levels in normal Boran steers for at least two weeks after intramuscular injection with the drug at a dose of 3.5 mg/kg bw. The assay will be useful in monitoring diminazene use, and development of resistance in trypanosomosis endemic areas.