Economic assessment of the performance of trypanotolerant cattle breeds in a pastoral production system in Kenya.

Cattle are the major source of food security and income for pastoral farmers in sub-Saharan Africa. However, infectious and parasitic diseases remain a major constraint to improved cattle productivity in the region. The use of animal health economics to support decision-making on cost-effective disease control options is increasingly becoming important in the developing world. Trypanotolerant indigenous Orma/zebu cattle in a trypanosomosis-endemic area of Kenya were evaluated for economic performance using gross-margin analysis and partial-farm budgeting. Orma/zebu and Sahiwal/zebu cross-bred cattle were exposed to similar husbandry practices and monitored for growth rate, incidence of common infections (trypanosomosis, anaplasmosis, babesiosis, East Coast Fever and helminthosis) and the cost of treatment assessed. Interview questionnaires were also used to assess the preference rating of the 2 breeds. Results indicated that incidence of infection was trypanosomosis 3%, anaplasmosis 58%, babesiosis 11%, East Coast Fever 22% and helminthosis 28%, with no significant difference between breeds. The Orma/zebu and Sahiwal/zebu breeds had comparable economic benefits, hence a pastoralist in Magadi division is likely to get similar returns from both breeds. This study therefore recommends adoption of not only the Sahiwal/zebu but also the Orma/zebu breed for cattle improvement in trypanosomosis endemic areas and conservation of indigenous genetic resources.

Efficacy of the novel diamidine compound 2,5-Bis(4-amidinophenyl)- furan-bis-O-Methlylamidoxime (Pafuramidine, DB289) against Trypanosoma brucei rhodesiense infection in vervet monkeys after oral administration.

Owing to the lack of oral drugs for human African trypanosomiasis, patients have to be hospitalized for 10 to 30 days to facilitate treatment with parenterally administered medicines. The efficacy of a novel orally administered prodrug, 2,5-bis(4-amidinophenyl)-furan-bis-O-methlylamidoxime (pafuramidine, DB289), was tested in the vervet monkey (Chlorocebus [Cercopithecus] aethiops) model of sleeping sickness. Five groups of three animals each were infected intravenously with 10(4) Trypanosoma brucei rhodesiense KETRI 2537 cells. On the seventh day postinfection (p.i.) in an early-stage infection, animals in groups 1, 2, and 3 were treated orally with pafuramidine at dose rates of 1, 3, or 10 mg/kg of body weight, respectively, for five consecutive days. The animals in groups 4 and 5 were treated with 10 mg/kg for 10 consecutive days starting on the 14th day p.i. (group 4) or on the 28th day p.i. (group 5), when these animals were in the late stage of the disease. In the groups treated in the early stage, 10 mg/kg of pafuramidine completely cured all three monkeys, whereas lower doses of 3 mg/kg and 1 mg/kg cured only one of three and zero of three monkeys, respectively. Treatment of late-stage infections resulted in cure rates of one of three (group 4) and zero of three (group 5) monkeys. These studies demonstrated that pafuramidine was orally active in monkeys with early-stage T. brucei rhodesiense infections at dose rates above 3 mg/kg for 5 days. It was also evident that the drug attained only minimal efficacy against late-stage infections, indicating the limited ability of the molecule to cross the blood-brain barrier. This study has shown that oral diamidines have potential for the treatment of early-stage sleeping sickness.

African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA.

Control of human African trypanosomiasis (HAT) is dependent on accurate diagnosis and treatment of infected patients. However, sensitivities of tests in routine use are unsatisfactory, due to the characteristically low parasitaemias in naturally infected individuals. We have identified a conserved sequence in the repetitive insertion mobile element (RIME) of the sub-genus Trypanozoon and used it to design primers for a highly specific loop-mediated isothermal amplification (LAMP) test. The test was used to analyse Trypanozoon isolates and clinical samples from HAT patients. The RIME LAMP assay was performed at 62 degrees C using real-time PCR and a water bath. DNA amplification was detectable within 25min. All positive samples detected by gel electrophoresis or in real-time using SYTO-9 fluorescence dye could also be detected visually by addition of SYBR Green I to the product. The amplicon was unequivocally confirmed through restriction enzyme NdeI digestion, analysis of melt curves and sequencing. The analytical sensitivity of the RIME LAMP assay was equivalent to 0.001 trypanosomes/ml while that of classical PCR tests ranged from 0.1 to 1000 trypanosomes/ml. LAMP detected all 75 Trypanozoon isolates while TBR1 and two primers (specific for sub-genus Trypanozoon) showed a sensitivity of 86.9%. The SRA gene PCR detected 21 out of 40 Trypanosoma brucei rhodesiense isolates while Trypanosoma gambiense-specific glycoprotein primers (TgsGP) detected 11 out of 13 T. b. gambiense isolates. Using clinical samples, the LAMP test detected parasite DNA in 18 out of 20 samples which included using supernatant prepared from boiled blood, CSF and direct native serum. The sensitivity and reproducibility of the LAMP assay coupled with the ability to detect the results visually without the need for sophisticated equipment indicate that the technique has strong potential for detection of HAT in clinical settings. Since the LAMP test shows a high tolerance to different biological substances, determination of the appropriate protocols for processing the template to make it a user-friendly technique, prior to large scale evaluation, is needed.

Detection of Trypanosoma evansi in camels using PCR and CATT/T. evansi tests in Kenya.

Camel trypanosomosis (Surra) causes high morbidity and is an impediment to the camel husbandry in Kenya. The lack of a sensitive diagnostic test has hindered the collection of accurate epidemiological data and institution of control programmes. A cross-sectional study was conducted in three districts of Kenya to estimate the prevalence of Trypanosoma evansi (T. evansi) and to compare four diagnostic tests: polymerase chain reaction (PCR), card agglutination test (CATT/T. evansi), microhaematocrit centrifugation technique (MHCT) and mouse inoculation (MI). A total of 549 camels were randomly sampled. The overall prevalence of Surra was 5.3% using MHCT, 26.6% using PCR and 45.9% using CATT/T.evansi. There was a significant difference (P < 0.001) between PCR and CATT/T.evansi test, MHCT and MI in detection of T. evansi. The prevalence of T. evansi was 39.8% in Samburu, 24.7% in Nanyuki and 14.4% in Isiolo districts using PCR. A male camel was 2.6 times more likely to be infected with T. evansi compared to a female camel (OR = 3.0% CI: 1.6, 4.1), while an adult camel was 2.2 times more likely to be infected compared to non-adults (OR = 2.2; 95% CI: 1.2, 5.0). There was a poor association between the presence of the published clinical signs and seropositivity (kappa = 0.12), PCR (kappa = 0.11) and MHCT (kappa = 0.05). However, there was a higher agreement between farmers' classification of disease with the PCR test (kappa = 0.5, n = 61). The mean PCV varied with age, presence of infection, locality and gender, with the lowest mean PCV being recorded in MHCT-positive animals (20.97 +/- 0.5) and from infected calves (19.5 +/- 1.2). This study shows that PCR was more sensitive in detecting T. evansi than other tests used. Further, the prevalence of T. evansi in the camel herds sampled is higher than that previously reported in Kenya, and that the judgment by camel keepers may be a reliable "pen-side" diagnostic test for Surra. Considering the low sensitivity of parasitological techniques in detection of chronic T. evansi infection and high cost of PCR, development of a sensitive pen side diagnostic test, with a low cost is still a priority.

Bowel function following primary repair of anorectal malformations at Kenyatta National Hospital.

OBJECTIVES: To evaluate bowel function following primary repair of anorectal malformation. DESIGN: A ten-year retrospective study. SETTING: Kenyatta National Hospital, Nairobi, Kenya. SUBJECTS: All patients with anorectal malformations attended to at Kenyatta National Hospital (KNH) within the study period who had posterior sagittal repair as a primary definitive procedure. All the children were over three years of age, toilet trained, and had their colostomies closed with an adaptation period of at least six months. RESULTS: Posterior sagittal repair was used to repair anorectal malformations in 352 patients. One hundred and ninety three patients were evaluated. Overall voluntary bowel movement (VBM) was achieved in 71.5% of the patients, soiling was present in 21.2% of the patients and constipation in 7.3% of the patients. More than seventy nine per cent of children who had their colostomy fashioned before the age of one month achieved VBM, while 61.1% of the patients achieved VBM when the colostomy was fashioned after five years. Overall, 77.0% of the females achieved VBM compared to 63.8% of males. Patients with a perineal fistula achieved VBM in 79.1% of males and 75.0% of females, 76.0% with vestibular fistula, 73.9% with a recto-urethral fistula, 56.0% of anorectal anomalies without a fistula, 25.0% of vaginal fistulae and 12.5% in vesical fistulae. Overall patients with sacral defects achieved VBM in 25.9% compared to 78.9% in patients with a normal sacrum. The patients with low anomalies achieved VBM in 75.4% compared to 46.1% with high anomalies. CONCLUSIONS: Posterior sagittal repair has been used to repair all anorectal malformations and has improved the quality of life of our patients, with better functional results expected in female patients, early colostomy fashioning and definitive repair, low or simple anomalies, and absence of sacral defects. The repair was associated with low morbidity and mortality.

Growth and mortality in sheep and goats under high tsetse challenge in Kenya.

Trypanosomosis is a major impediment to livestock production and economic development in those areas of Africa where it is endemic. Although small ruminants appear to perform better than cattle in various agro-ecological zones, the importance of trypanosomosis has not been extensively investigated in these livestock. This study was designed to investigate the prevalence of trypanosomosis in sheep and goats in an endemic area and to evaluate the performance of different breeds under high tsetse challenge and the potential role of chemoprophylaxis in the control of the disease. The results showed that tsetse flies feed readily on small ruminants, and that these animals are susceptible to trypanosomosis. The Small East African goats acquired fewer infections than the Black Head Persian and Dorper sheep used in the study. In both sheep and goats, chemoprophylaxis with isometamidium chloride (Samorin, Rhone Merieux, Annecy, France) was protective, resulting in fewer infections and higher body weight gain. Trypanosomosis caused anaemia in both sheep and goats, and animals whose PCV fell below 15% rarely recovered, even with trypanocidal drug treatment. The peak transmission period was between 1 and 3 months after the peak tsetse fly density, which raises the possibility of effective strategic prophylaxis.

Unravelling the phylogenetic relationships of African trypanosomes of suids.

African trypanosomes of the subgenera Nannomonas and Pycnomonas have been recorded from both wild and domestic suids. However, complete descriptions of some of these trypanosomes with regard to host range, pathogenicity, transmission and distribution are still lacking. Neither the recently described Trypanosoma (Nannomonas) godfreyi nor Trypanosoma (Nannomonas) congolense Tsavo have been isolated from mammalian hosts, while Trypanosoma (Pycnomonas) suis remains the rarest of the Salivarian trypanosomes. The only isolate presumed to be of the latter species is maintained at the Kenya Trypanosomiasis Research Institute, Nairobi. We present here the results of characterization of this isolate by morphology, tsetse transmission, the use of species-specific DNA probes and DNA sequence analysis. Morphology in stained blood smears revealed a small trypanosome with a free flagellum. Experimental transmission through Glossina morsitans morsitans showed a developmental cycle typical of subgenus Nannomonas A positive identification was obtained with species-specific PCR primers for T. congolense Tsavo; moreover, the sequence of the SSU rRNA gene was almost identical to that of T. congolense Tsavo on database. In phylogenetic analysis of the SSU rRNA genes of Salivarian trypanosomes, T. congolense Tsavo grouped with T. simiae rather than T. congolense, suggesting that the name T. simiae Tsavo is more appropriate.

Standardised tests in mice and cattle for the detection of drug resistance in tsetse-transmitted trypanosomes of African domestic cattle.

Resistance to the drugs used to control African animal trypanosomosis is increasingly recognised as a constraint to livestock production in sub-Saharan Africa. The most commonly used tests for detection of trypanocidal drug resistance are tests using mice or ruminants, but these suffer from lack of standardisation and hence it may be difficult to compare the results of different investigators. Tests in mice are less expensive than tests in ruminants, but while tests in mice they may be useful as a general guide to resistance in a geographic area they should not be extrapolated to cattle on an individual trypanosome level. Moreover, the commonly used protocols are too laborious for their application to large number of trypanosome isolates on an area-wide basis. This paper presents guidelines for standardised testing of trypanocidal drugs in vivo, and introduces a simplified single-dose test for use in mice, which is convenient for use in areas with limited laboratory facilities. The single-dose test is appropriate for characterisation of geographic areas in terms of trypanocidal drug resistance using large numbers of trypanosome isolates, for making comparisons between areas, and for monitoring changes in trypanocidal drug resistance over time. Multiple-dose tests may be used to determine the degree of resistance of individual stabilates to be determined precisely in mice are also described, but for logistical reasons these will rarely be conducted on more than a few stabilates, and testing of a larger number of stabilates in the single-dose test will generally provide more useful information. Finally, we describe tests in cattle that may be used to determine the efficacy of recommended curative doses of trypanocidal drugs for the treatment of infection with individual trypanosome isolates, including Trypanosoma vivax, which is rarely infective for mice.

Comparative sensitivity of dot-ELISA, PCR and dissection method for the detection of trypanosome infections in tsetse flies (Diptera: glossinidae).

A visually read dot-enzyme linked immunosorbent assay (dot-ELISA) developed for the detection of trypanosomes in tsetse flies (Glossina spp.) was evaluated in the laboratory and under field conditions. In the evaluation, the fly dissection method was used as a standard technique and compared to the polymerase chain reaction (PCR). In laboratory studies, 133 and 126 tsetse flies were experimentally infected with different stocks of Trypanosoma brucei and T. congolense, respectively. Twenty-five days after infection, the flies were dissected and tested for the presence of trypanosomes using dot-ELISA and PCR. Dot-ELISA detected 98.4% of T. brucei and 94% of T. congolense infections in tsetse midguts, while PCR detected 97.6% of T. brucei and 96% of T. congolense tsetse midgut samples. For field evaluation of dot-ELISA, 700 tsetse flies were caught and screened for trypanosome infections by dissection. Seven of these (1%) had trypomastigotes in the midgut, 23 (3.3%) in the proboscis and none had trypanosomes in the salivary glands. All the flies with midgut infections also had trypanosomes in their proboscides. Five of the seven flies (71.4%) with midgut infections revealed by dissection, were also positive for T. congolense by the dot-ELISA and PCR techniques. Dot-ELISA detected T. congolense infections in an additional 86 (12.4%) of the 700 flies dissected. Of the 23 infections in the proboscis, 16 were T. vivax. Dot-ELISA detected 13 of the 16 (81%) while PCR detected 15 of 16 (94%) T. vivax infections. No T. brucei infection was detected by any of the methods in all the 700 tsetse flies examined. The results obtained from both the laboratory and field studies indicate that the dot-ELISA and PCR techniques are sensitive and species-specific in revealing trypanosome infections in tsetse flies. While dot-ELISA required a single test to detect T. congolense, several primer pairs were needed for PCR. The potential use of dot-ELISA as a tool for studying the epidemiology of trypanosomosis, while considering its field applicability and relatively lower cost is discussed.

Application of the Vettest 8008 system for the biochemical analysis of vervet monkey plasma.

A clinical biochemistry analyser designed specifically for veterinary use was used to analyse plasma samples from 24 vervet monkeys (Cercopithecus aethiops). Two millilitres of heparinised blood was collected from each of the 24 monkeys on four occasions at intervals of one week. Plasma was separated and analysed for the concentrations of triglycerides, cholesterol, total proteins, albumin, globulins, creatinine and blood urea nitrogen (BUN) and the activities of alkaline phosphatase (AP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK). The tests were easy to perform, used small volumes of plasma, and yielded consistent results for most of the analytes. The activities of CK and AP, but not AST, appeared to be influenced by haemolysis, and there were significant individual variations in the activity of LDH.

Financial analysis of animal trypanosomiasis control using cypermethrin pour-on in Kenya.

The financial impact of use of cypermethrin pour-on (Ectopor(R)) in control of animal trypanosomiosis was determined in a trial undertaken by the Kenya Trypanosomiasis Research Institute (KETRI). This trial started in December 1990 and ended in February 1992. It was undertaken in two adjacent ranches in the coast province of Kenya. The trial site was in an area of high apparent density (AD) of tsetse flies, and at the start of the trial no cattle were kept in this area. Cypermethrin was applied fortnightly to the 1100 steers which were kept in pour-on ranch 'A' while another 100 steers were kept in control ranch 'B' to act as control sentinels. From the main pour-on group, 100 animals were identified as the pour-on sentinels and compared to the control sentinels which received no pour-on.Pour-on application led to a significant decrease in the tsetse AD in the pour-on ranch A to 90% of the initial AD in some areas. The animals treated with pour-on had a significantly higher mean packed-cell volume (PCV). The weekly prevalence of trypanosome infections in animals treated with pour-on was <4% with only one exception when it was <10%. In the control animals, the prevalence ranged between 10 and 50% (with a few exceptions when it was <10%). The incidence of tick-borne diseases was lower in the pour-on animals. The mean monthly weights of the pour-on animals was significantly higher, and at the end of the trial the pour-on animals had a mean weight gain of 136.70+/-16.7kg while the control animals had gained 97.16+/-22.6kg. The financial net return of using cypermethrin pour-on was positive and the financial rate of return of 122.6% indicated that use of the pour-on was highly beneficial despite the high cost of the product.

Some pharmacokinetic parameters of the trypanocidal drug homidium bromide in Friesian and Boran steers using an enzyme-linked immunosorbent assay (ELISA).

Pharmacokinetic studies on the trypanocidal drug homidium bromide using a competitive enzyme immunoassay (detection limit 0.1 ng/mL) are reported for non-infected Friesian and Boran steers following treatment with homidium bromide at a dose of 1.0 mg/kg b.w. Following intravenous (i.v.) treatment of Friesian steers (n = 5), the mean serum drug concentrations were 31.9 +/- 2.1 and 3.9 +/- 0.4 ng/mL at 1 and 24 h, respectively. The decline in serum drug concentration was tri-exponential with half-lives of 0.064 +/- 0.037 h for t1/2 alpha, 7.17 +/- 1.87 h for t1/2 beta and 106.3 +/- 6.6 h for t1/2 gamma for distribution and elimination phases 1 and 2, respectively. Drug was detectable in serum for 17 days following treatment. The mean residence time (MRT) was 63.4 +/- 7.5 h. Following intramuscular (i.m.) treatment of Friesian steers (n = 5), the drug concentration at 1 h after treatment was 72.5 +/- 2.2 ng/mL. This declined to 9.8 +/- 1.8 ng/mL at 24 h. Low concentrations of between 0.1 and 0.3 ng/mL remained in circulation for up to 90 days post-treatment. Following intramuscular treatment of Boran steers (n = 5), the mean serum drug concentration at 1 h after treatment was 112.1 +/- 40.3 ng/mL. By 24 h after treatment, the concentration had fallen to 13.0 +/- 3.3 ng/mL. Thereafter, the serum drug concentration-versus-time profile and the pharmacokinetic parameters obtained following non-compartmental analysis were similar to those obtained following intramuscular treatment of Friesian steers.

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the determination of the trypanocidal drug homidium in serum of treated cattle.

Two enzyme-linked immunosorbent assays (ELISA) for the determination of homidium in serum of treated cattle have been developed and evaluated. One is a direct competition (Assay 1) and the other an indirect competition assay (Assay 2). Both assays are highly sensitive with a limit of detection of 0.1 ng homidium per mL serum. Homidium levels were measurable in serum of cattle for over 2 months following administration of a single intramuscular (i.m.) dose at 1 mg/kg bodyweight. The level of sensitivity afforded by these assays makes them potentially useful tools in the pharmacokinetic evaluation of homidium and for investigating drug resistance or causes of drug failure. Assay 2 was chosen as being most suitable for further studies.

Nitric oxide production in vervet monkeys (Cercopithecus aethiops) infected with Trypanosoma brucei.

A retrospective study of nitrate concentration in serum and cerebrospinal fluid (CSF) from vervet monkeys (Cercopithecus aethiops) infected with Trypanosoma brucei was undertaken. Serum nitrate was significantly elevated in parasitaemic animals. CSF nitrate detection correlated with the presence of parasites in the CNS. The results provide evidence for the production of nitric oxide (NO) in response to infection in a primate model of human African trypanosomiasis and provide the basis for the use of such a model in studies of the immunopathological effects of NO in human trypanosomiasis.

Studies on host resistance to tick infestations among trypanotolerant Bos indicus cattle breeds in east Africa.

Recent epidemiological studies carried out in East Africa have indicated that some Bos indicus cattle breeds such as the Orma Boran and Maasai Zebu have a degree of trypanotolerance worth exploitation by their introduction into trypanosomosis endemic areas where other cattle breeds cannot survive. However, in most areas of East Africa, trypanosomosis, ticks, and tick-borne diseases occur together. It is therefore important to obtain information on the susceptibility of these breeds to tick infestation and tick-borne diseases. This study was therefore designed to determine the susceptibility of these cattle breeds to tick infestations. They were compared with the Galana Boran (trypanosusceptible) and the Friesian (susceptible to tick infestations, tick-borne diseases, and trypanosomosis). The four breeds of cattle were exposed to natural tick challenge for a period of seven months and whole body weekly tick counts were done on each animal. Significant differences to tick infestations among the four breeds were observed. For both Rhipicephalus appendiculatus and Boophilus decoloratus, susceptibility to infestation increased in the order, Maasai Zebu, Orma Boran, Galana Boran and Friesian. The results generated by this pilot study so far suggest that variation in susceptibility to tick infestations exists among the four breeds. The Orma Boran and Maasai Zebu showed greater resistance to tick-infestations than the Galana Boran and Friesian. This suggests that utilization of these trypanotolerant cattle breeds could be feasible even in the face of tick challenge and should therefore be considered when planning integrated trypanosomosis and tick control strategies.

Changes in atrial natriuretic factor and plasma renin activity in dogs infected with Trypanosoma brucei.

When beagle dogs were infected with Trypanosoma brucei, a marked reduction in the plasma concentration of atrial natriuretic factor (ANF) occurred in the terminal stage of the disease during weeks 3 and 4. At the same time there was an increase in plasma renin activity (PRA) after infection. Ultrastructural studies of the atria of these dogs demonstrated a reduction in ANF granules. The changes in ANF and PRA occurred in association with severe pancarditis and the development of heart failure. By impairing the ability of the heart and kidneys to regulate blood volume, the alterations in ANF and PRA could be involved in the pathogenesis of heart failure in T. brucei-infected dogs.

Elevation of the concentration of acute phase proteins in dogs infected with Trypanosoma brucei.

Plasma concentrations of the acute phase proteins (APP), C-reactive protein (CRP) and haptoglobin (Hp), increased markedly following experimental infection of dogs with Trypanosoma brucei. The highest concentrations of CRP were observed immediately after peaks of parasitaemia. Treatment with curative doses of the trypanocidal drug suramin caused a rapid decrease in CRP. Relapse infections after subcurative treatment were followed by a reappearance of high plasma CRP concentrations. Haptoglobin remained elevated during the course of the disease. Curative treatment with suramin caused a gradual but slow decrease in Hp while subcurative treatment caused no significant changes. Thus, the estimation of CRP was useful in determining the presence of active infection and the success of chemotherapy. High Hp levels in severely anaemic dogs indicated that intravascular haemolysis does not contribute significantly to the anaemia associated with T. brucei infections in dogs. These conclusions need confirmation from a larger experiment.