Immunosuppression in bovine trypanosomiasis: studies with louping-ill vaccine.

The antibody response to louping-ill virus vaccine was examined in mice infected with Trypanosoma brucei and T congolense, and in Ethiopian cattle experimentally infected with T brucei, T congolense and T vivax. In mice the antibody response was completely suppressed, while in cattle infected with T congolense and T vivax the antibody response to the vaccine was only 10 per cent that of uninfected animals. In contrast, the response of cattle infected with T brucei was not significantly reduced, and this was attributed to their relatively light and transient parasitaemias. Trypanocidal chemotherapy (diminazine aceturate) administered on the same day as vaccination largely restored the competence of the immune response of both mice and cattle infected with T congolense. The use of such drugs should be considered when cattle are vaccinated in trypanosome endemic areas.     

Drug-resistant Trypanosoma congolense in naturally infected donkeys in north Omo Zone, southern Ethiopia

A three-part study was conducted to determine the efficacy of isometamidium chloride in donkey populations naturally infected with trypanosomes in north Omo Zone, southern Ethiopia. In the first, 373 randomly selected donkeys from four villages were examined for trypanosome infections by the dark ground/phase contrast buffy coat technique (BCT) in November 1999. The trypanosome prevalence was 18.2% (95% confidence interval (CI): 14.4, 22.5) and Trypanosoma congolense was the most common species accounting for 66.2% of the overall infections. In the second part, 40 infected donkeys were selected and treated with a prophylactic dose of 1.0mg/kg of isometamidium chloride and thereafter monitored every 14 days for 90 days. Trypanosomes were detected in eight donkeys within 1 month and in 20 donkeys within 2 months of treatment. About 16% (5/32) of donkeys infected with T. congolense were detected parasitemic 1 month after treatment. In addition, the result also revealed that all relapse/breakthrough infections were due to T. congolense. In the third part of this study mice were infected with two T. congolense field isolates from donkeys that were found to be parasitemic within 1 or 2 months after isometamidium treatment. The mice were treated with ranges of doses of isometamidium chloride or diminazene aceturate and thereafter followed for relapse infection. Isometamidium chloride at doses 0.5-4 mg/kg body weight and diminazene aceturate at doses of 3.5-28 mg/kg body weight failed completely to cure T. congolense infections in any of the mice.     

Application of PCR and DNA probes in the characterisation of trypanosomes in the blood of cattle in farms in Morogoro, Tanzania

Polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) probes were used to characterise trypanosomes from cattle in Morogoro region of Tanzania. Blood samples collected from 390 beef and dairy cattle in selected farms in Morogoro region were examined for presence of trypanosomes using the buffy coat technique (BCT) and blood smears (BSs). Fifty-two animals were found infected: 40 with Trypanosoma congolense, 10 with T. vivax and two with both T. congolense and T. vivax. DNA extracted from all the parasitologically positive and 62 randomly selected parasitologically negative samples were subjected to PCR amplification using primers specific for different trypanosome species. Using a set of seven specific-pairs of primers on the parasitologically positive samples, we detected only T. congolense, either the Savannah- or the Kilifi-type, as single or mixed infections. With the PCR, trypanosome DNA could be detected in 27 (43%) out of 62 samples that were parasitologically negative. DNA hybridisation using probes specific for Savannah- or Kilifi-types T. congolense, or T. vivax, confirmed the presence of these parasites in cattle kept on some farms in Morogoro region of Tanzania. From these studies, it is clear that there is a need to undertake molecular epidemiological studies to determine the distribution of trypanosome species and subspecies, and to assess the economic impact of these parasites in the productivity of livestock in Tanzania. In particular, it would be desirable to verify the assumed association between the different presentations of trypanosomosis on one hand and genotypes of T. congolense on the other.     

Analysis of host genetic factors influencing African trypanosome species infection in a cohort of Tanzanian Bos indicus cattle

Trypanosomosis caused by infection with protozoan parasites of the genus Trypanosoma is a major health constraint to cattle production in many African countries. One hundred and seventy one Bos indicus cattle from traditional pastoral Maasai (87) and more intensively managed Boran (84) animals in Tanzania were screened by PCR for the presence of African animal trypanosomes (Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei), using blood samples archived on FTA cards. All cattle screened for trypanosomes were also genotyped at the highly polymorphic major histocompatibility complex (MHC) class II DRB3 locus to investigate possible associations between host MHC and trypanosome infection. Overall, 23.4% of the 171 cattle tested positive for at least one of the three trypanosome species. The prevalence of individual trypanosome species was 8.8% (T. congolense), 4.7% (T. vivax) and 15.8% (T. brucei). The high prevalence of T. brucei compared with T. congolense and T. vivax was unexpected as this species has previously been considered to be of lesser importance in terms of African bovine trypanosomosis. Significantly higher numbers of Maasai cattle were infected with T. brucei (23.0%, p=0.009) and T. congolense (13.8%, p=0.019) compared with Boran cattle (8.3% and 3.6%, respectively). Analysis of BoLA-DRB3 diversity in this cohort identified extensive allelic diversity. Thirty-three BoLA-DRB3 PCR-RFLP defined alleles were identified. One allele (DRB3*15) was significantly associated with an increased risk (odds ratio, OR=2.71, p=0.034) of T. brucei infection and three alleles (DRB3*35, *16 and *23) were associated with increased risk of T. congolense infection. While further work is required to dissect the role of these alleles in susceptibility to T. brucei and T. congolense infections, this study demonstrates the utility of FTA archived blood samples in combined molecular analyses of both host and pathogen.     

The interaction of Trypanosoma congolense and Haemonchus contortus infections in trypanotolerant N'Dama cattle.

The interactions between Trypanosoma congolense and Haemonchus contortus infections were studied in N'Dama calves. A total of 38 N'Dama bulls was divided into four groups and each group infected either with H. contortus 1 week after infection with T. congolense or with T. congolense 4 weeks after infection with H. contortus, or with either infection singly. Parasitological (faecal egg counts, parasitaemia), haematological (packed cell volume, white blood cell counts, albumin) and clinical parameters (body weight change, mortality rate) were compared among the various groups. The results showed a reduced prepatent period and a markedly increased pathogenicity of H. contortus infections in animals with a concurrent T. congolense infection. The most harmful combination was a H. contortus infection 1 week after the T. congolense infection which resulted in a progressive and severe anaemia, accompanied by hypoalbuminaemia, increased weight loss and high mortality. The anaemia induced by dual infections showed a low responsiveness to chemotherapy and in several cases supportive treatment did not help recovery. The results also showed that animals with a concurrent T. congolense and H. contortus infection ran a higher risk of succumbing during the infection, and also during 10 weeks following treatment. Although infections with T. congolense alone produced no clinical signs, they were found to significantly reduce the ability of infected animals to mount a normal response to a subsequent H. contortus infection. It was concluded that the increased H. contortus egg excretion observed in animals infected with both parasites might significantly increase the risk of nematode infections and that the reduced prepatent period might necessitate more frequent anthelmintic treatments. These interactions should, therefore, be considered wherever attempts are made to control these two diseases.     

The transmissibility of Trypanosoma congolense seems to be associated with its level of resistance to isometamidium chloride

In large parts of Africa the control of livestock trypanosomiasis relies on the use of trypanocidal drugs. Resistance against the available compounds is developing rapidly in the trypanosome population. The effect of the development of drug resistance on the fitness of the trypanosome is not well known. To determine the effect of the development of resistance to isometamidium chloride on the trypanosome's transmissibility, transmission experiments were conducted. Use was made of three isogenic clones of Trypanosoma congolense with different susceptibility to the drug. The infection rate in Glossina morsitans morsitans differed significantly between clones and was significantly higher in tsetse flies infected with the T. congolense clone with the highest level of drug resistance.     

Trypanosome infection rate in cattle at Nguruman, Kenya

Trypanosome infection rate in cattle at Nguruman was investigated in a study conducted in 1984-1986. Shifting pastoralism significantly reduced trypanosome infections in cattle. The cattle were more heavily infected with Trypanosoma congolense (16.5%) than Trypanosoma vivax (4.95%) and Trypanosoma brucei (0.19%). Trypanosoma theileri was observed only once among the cattle examined. Mixed trypanosome infections in cattle were observed to be 2.75% and 0.014% for T. congolense/T. vivax and T. congolense/T. brucei, respectively. The duration of infection in the cattle was 55 days for T. congolense and 79 days for T. vivax. High infections in cattle were observed 2 months after the rains, which were concomitant with high tsetse densities.     

Susceptibility of West African Dwarf goats and WAD x Saanen crosses to experimental infection with Trypanosoma congolense

West African Dwarf goats (WADs) and their Saanen crosses were experimentally infected with Trypanosoma congolense. No significant differences were found between trypanosome parasitaemia and antibody response of the crossbred and WAD goats. Neither the WAD goats nor the Saanen crosses were able to control the drop in PCV following trypanosome infection. The level of anaemia caused by the trypanosome infection was similar in the two breeds during the trial. Based on these findings, no difference in tolerance or susceptibility to T. congolense could be demonstrated between the WAD goats and their Saanen crosses. Although the weight of all goats increased during the trial, the crosses gained significantly more weight than the WAD goats. The trypanosome infection reduced the growth rate of both breeds, but this reduction was not statistically significant. Crossbreeding trypanotolerant WADs with trypanosusceptible Saanen goats might, therefore, be an effective means of increasing productivity.     

Pathological changes in male genitalia of cattle infected with Trypanosoma vivax and Trypanosoma congolense

Samples for histological studies were taken from the genitalia of 14 bulls (five infected with Trypanosoma vivax, five with T. congolense and four uninfected control animals), slaughtered 12, 22 or 30 weeks post-infection. Infection with Y58 strain of T. vivax and strain 2295 of T. congolense caused various grades of lesions in the male reproductive organs, especially the testes and epididymides. T. congolense produced more severe degenerative changes than T. vivax. It is concluded that in long-standing cases, the result of trypanosome infection is either serious infertility or even sterility.     

Isometamidium chloride prophylaxis against Trypanosoma congolense challenge and the development of immune responses in Boran cattle

Twenty-four Boran cattle were injected with isometamidium chloride (1 mg/kg bodyweight) to investigate the duration of drug-induced prophylaxis against infection by metacyclic forms of Trypanosoma congolense and to determine if specific antibody responses to the organism were mounted by animals under chemoprophylactic cover. Complete protection against either single challenge by five tsetse flies infected with T congolense, or repeated challenge at monthly intervals by five tsetse flies, lasted for five months. Six months after treatment, two-thirds of the cattle were resistant to challenge, irrespective of whether subjected to single or multiple challenge with trypanosome-infected tsetse flies, or titrated doses of in vitro-cultured metacyclic forms of T congolense (5 X 10(2) to 5 X 10(5) organisms), inoculated intradermally. No animal which resisted infection developed detectable skin reactions at the site of deposition of metacyclic trypanosomes or produced trypanosome-specific antibodies. It was concluded that drug residues effectively limited trypanosome multiplication at the site of deposition in the skin, thus preventing subsequent parasitaemia or priming of the host's immune response.     

Interference in the establishment of tsetse-transmitted Trypanosoma congolense, T. brucei or T. vivax superinfections in goats already infected with T. congolense or T. vivax

An interference phenomenon that delays superinfection with a trypanosome species different from that used for the initial infection has been found to occur in goats. Following tsetse transmission of Trypanosoma brucei to goats already infected with T. congolense, there was a delay in chancre development, as well as in the appearance of T. brucei and anti-T. brucei antibodies in the blood when compared to previously uninfected goats. However, there was no delay in the establishment of a tsetse-transmitted superinfection with T. vivax in goats already infected with either T. congolense or in animals already infected with a different serodeme of T. vivax.     

Enhanced protection against Heligmosomoides polygyrus in IL-2 receptor β-chain overexpressed transgenic mice with intestinal mastocytosis

IL-2 receptor β-chain overexpressed transgenic (Tg2Rβ) mice lack NK cells, but the development of other lymphocyte subsets and macrophages remained apparently intact. These mice also exhibit intestinal mastocytosis. Helminth infection induces various immune responses, such as mast cells, goblet cells, eosinophils and IgE, mediated by Th2 cytokines. IL-4 is also important in the regulation of resistance and susceptibility to Heligmosomoides polygyrus infection. However, there are contradictory results about the relation between resistance to H. polygyrus and intestinal mastocytosis. The present study showed that Tg2Rβ mice suppressed worm fecundity with mastocytosis without an increase of the levels of goblet cells, eosinophils and IgE compared with control mice. These results clearly indicated that mast cells have the ability for to protect against H. polygyrus infection. However, additional studies are required to evaluate protective effector mechanisms against H. polygyrus.     

Performance of enzyme-linked immunosorbent assays for detection of antibodies against T. congolense and T. vivax in goats

Indirect ELISAs using denatured antigen preparations of Trypanosoma (T.) congolense (TcAGd) and T. vivax (TvAGd) for detection of anti-trypanosome antibodies in bovine serum (I-TAB ELISAs), were adapted for serodiagnosis in goats. The diagnostic proficiency, the cross-reactivity with sera from heterologous trypanosome infections and the operational performance of the assays were evaluated on experimentally trypanosome-infected goats. The I-TAB ELISA (TcAGd) detected antibodies in all T. congolense infected goats (100% overall sensitivity) from 2 to 4 weeks post-infection (p.i.) until the end of the experiments. Specificity tested on 92 uninfected goats was 96.7%. Extensive cross-reactions of I-TAB ELISA (TcAGd) with sera from T. vivax or T. brucei infected goats were observed. The I-TAB ELISA (TvAGd) detected antibodies in 5 of the 6 T. vivax infected goats, specificity tested on uninfected goats was 100%. Cross-reactivity with sera from T. congolense or T. brucei infected goats remained limited. Infecting species identification based on the highest percent positivity (PP) in both systems, correctly identified all T. congolense infections, but misidentified in 2/19 occasions a T. vivax infection as a T. congolense infection. In the absence of T. brucei specific antigen coated plates, T. brucei infections were identified in, respectively, 7/9 and 2/9 occasions as T. congolense or T. vivax infections. Acceptable inter-plate repeatability was observed. The implications of results and technical requirements for ongoing applied research are discussed.     

Prevalence of bovine trypanosomosis and trypanocidal drug sensitivity studies on Trypanosoma congolense in Wolyta and Dawero zones of southern Ethiopia

Cross-sectional studies were conducted in tsetse and non-tsetse-controlled areas of the Southern Nation Nationalities and Peoples Regional State (SNNPRS) of Ethiopia to determine the prevalence of bovine trypanosomosis as well as drug sensitivity tests on Trypanosoma congolense in both naturally and experimentally infected cattle and mice, respectively. A total trypanosome prevalence of 4.8% (95% CI: 1.8-7.5) and 20.4% (95% CI: 14-26.8) were recorded in the tsetse-controlled study area of Humbo district and the non-tsetse-controlled area of Mareka district, respectively, indicated statistically significant difference between the two areas (P<0.001). The mean PCV value for Humbo and Mareka was 26.2 (95%: 25.7-26.7) and 22.7 (95% CI: 22.1-23.3), respectively, which were also statistically significant (P<0.001). The prophylactic activity of isometamidium chloride (ISMM) was observed in Humbo on nine naturally positive zebu cattle. Breakthrough infections were recorded in (6/9) 66.7% of the cases in less than 5 weeks. A qualitative assay on mice was conducted on two T. congolense isolates obtained from the breakthrough cases with ranges of doses of ISMM and diminazene diaceturate (DA). Thereafter the mice were followed for relapse infection. ISMM at doses 0.5-4 mg/kg body weight (bw) and DA at doses of 3.5-28 mg/kg bw failed completely to cure T. congolense infections in any of the mice. A quantitative assay on mice was conducted on four T. congolense isolates obtained from Mareka. The four isolates were pooled into two pools (Pool-1 and Pool-2) for the quantitative assay on mice. The pooled isolates were tested with the same trypanocidal drugs and ranges of doses as it was used for the qualitative assay on mice. The minimum curative dose (MCD) of ISMM that cleared T. congolense infected mice was 4 and 2mg/kg bw for Pool-1 and Pool-2, respectively, whereas MCD of DA was 28 and 14 mg/kg bw, in Pool-1 and Pool-2, respectively. Although cloned populations were not used to prove whether the observed resistance was at the individual level or not, the results show that there is resistance to both ISMM and DA; failure of the "sanative pair".     

The intensity of resistance by mature Merino ewes against Haemonchus contortus and Tichostrongylus colubriformis in single-species and combined-species infection

Three-year-old, non-lactating and non-pregnant Merino ewes, raised on pasture under a program of strategic treatment with anthelmintic and found to be extremely resistant to "trickle" infection with Haemonchus contortus, were given single-dose infections with either H. contortus or Trichostrongylus colubriformis or both species together. The purpose was to ascertain the intensity of protective immunity against the 2 parasites in sheep with immunity acquired from a presumably slight exposure to infection. To provide a criterion, some infected ewes were immunosuppressed with corticosteroid, dexamethasone. Untreated ewes were extremely resistant to challenge infection with either 15,000 or 150,000 H. contortus or 15,000 T. colubriformis. Surprisingly, when mixed infection was given, egg counts for H. contortus were significantly elevated compared with infection by that species alone. Antibody to antigens from infective larval and adult H. contortus was measured in serum by enzyme-linked-immunosorbent assay (ELISA) during the course of infection. Serum titres against larval antigens were significantly depressed when infections with either H. contortus or T. colubriformis were permitted by immunosuppression with dexamethasone, whereas those against adult antigen were depressed when infection with T. colubriformis was permitted.     

Protective efficacy of isometamidium chloride and diminazene aceturate against natural Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax infections in cattle under a suppressed tsetse population in Uganda

The protective efficacy of isometamidium chloride (ISMM) and diminazene aceturate (DIM) against Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax infections in cattle under a suppressed tsetse population was assessed in southeast Uganda. A total of 66 and 57 trypanosome-infected cattle were treated with ISMM and DIM, respectively together with 177 trypanosome-free animals not treated were followed for 12 months, checked every 4 weeks. There was no statistical difference in the mean time to infection with any trypanosome species in animals treated with ISMM or DIM. However, the mean time to trypanosome infection was significantly longer for treated animals than controls. The mean time to infection with each of the three trypanosome species differed significantly, with the average time to T. vivax infection the lowest, followed by T. congolense and then T. brucei. The protective efficacy of DIM was as good as that of ISMM; implying curative treatments against trypanosomosis are sufficient for combination with tsetse control. Isometamidium chloride or DIM had the highest impact on T. brucei and T. congolense infections in cattle.     

The transmission of mixed Trypanosoma brucei brucei/T. congolense infections by tsetse (Glossina morsitans morsitans)

Laboratory experiments and field observations clearly show that tsetse flies can be carriers of mixed trypanosome infections. Question remains how easy it is for the tsetse fly to acquire such a mixed infection during the first bloodmeal. This is of particular importance in the epidemiology of Trypanosoma brucei s.l., often a cryptic infection and difficult to transmit to non-teneral tsetse flies. To determine the transmission rate of T. brucei as part of a mixed infection, teneral Glossina morsitans morsitans were fed once on cattle with a mixed (Trypanosoma brucei brucei/Trypanosoma congolense) or single (T. brucei) infection. Of the 140 flies fed on animals with a mixed infection and examined 30 days later, 4 had a metacylic T. brucei infection, 29 a T. congolense infection and 13 a mixed T. brucei/T. congolense infection. There was no significant difference between the transmission rate of T. brucei as a single or as part of a mixed infection. The high proportion of mixed T.b. brucei/T. congolense infections was explained best by a model implying that if a fly is refractory to T. congolense, it is also refractory to T.b. brucei and vice versa. Hence, results suggest that the transmission of T.b. brucei is affected mainly by the vectorial capacity of flies and not by concurrent trypanosome infections in the host.     

Relationships between trypanosome infection measured by antigen detection enzyme immunoassays, anaemia and growth in trypanotolerant N'Dama cattle.

Relationships were evaluated between trypanosome infection as measured by antigen detection enzyme immunoassays (antigen ELISA), anaemia as determined by average packed red cell volume (PCV), and animal performance as assessed by daily weight gain in 99 N'Dama cattle in Gabon exposed to natural tsetse challenge at 11.5 months of age and recorded 14 times over a 13 week period. Approximately half the animals were found to be infected for an average of five of the 14 times that they were examined: 38% with Trypanosoma congolense, 13% with Trypanosoma vivax and 49% with a mixed infection. Trypanosoma congolense infections had significant deleterious effects on animal growth, while T. vivax infections did not. Animals found on several occasions to be infected with T. congolense had significantly lower PCV values than those demonstrated to be infected on fewer occasions. No relationship was found between mean optical density (OD) values in antigen ELISA and PCV values. Animals capable of maintaining PCV values, even when antigen ELISA positive on a high number of occasions, grew at the same rate as uninfected animals. Animals that could not maintain PCV values when infected had poorer growth. Antigen ELISA has the potential to increase the efficiency of selection of trypanotolerant N'Dama cattle under tsetse challenge in the field, in three main ways. (1) Accurate identification of trypanosome species, especially in mixed species infections, clarifies relations between infection, anaemia and animal performance. (2) Detection of animals antigenaemic without patent parasitaemia could allow individuals with superior ability to control trypanosome infection to be identified. (3) More accurate measurement of the proportion of time an animal is infected allows more accurate evaluation of its anaemia control capability.     

Development of Trypanosoma congolense, T vivax and T brucei in the skin reaction induced in goats by infected Glossina morsitans centralis: a light and electron microscopical study

The development and distribution of Trypanosoma congolense, T vivax and T brucei in the skin of goats was examined after the animals were bitten by infected Glossina morsitans centralis. Following the tsetse bite, the trypanosomes in the skin multiplied, reaching maximum numbers when the skin reaction (chancre) of the host attained its maximum size. In goats infected with T vivax and T brucei, trypanosomes were observed circulating in the blood before the peak of the chancre, while in T congolense-infected goats microscopically detectable parasites were found in blood only during the decline of the chancre. In contrast to T vivax, large numbers of T congolense and T brucei parasites were found in the skin following tsetse-transmitted infection. Ultrastructural differences were observed in T congolense and T brucei indicating an intracutaneous transformation from metacyclic to blood stream forms. T congolense forms in the skin reactions had a well developed secretory reticulum, small mitochondria and lacked large lipid inclusions compared to metacyclic and blood stream forms. The intracutaneous forms of T brucei had smaller mitochondria, the glycosomes were of more uniform size and the rough endoplasmic reticulum was less developed than in metacyclic or blood stream forms.