Atypical hyperpachymorph Trypanosoma (Nannomonas) congolense forest-type in a dog returning from Senegal.

Trypanosoma congolense forest-type was identified by PCR in France, in a dog returning from Senegal. This paper describes the morphological features of the parasite on Giemsa-stained smears. Slender forms and "latent bodies" represent 30.4% and 20.4%, respectively. Some rosettes have been observed (0.8%). The predominant form (48.4%) is stumpy, close to "montgomeryi-form", but it is unusually broad, with a width/length ratio (WLr) of 0.40-0.55, while that of "montgomeryi-forms" is close to 0.3. To the best of our knowledge, this is the first description of such a form of T. (Nannomonas). Also unusual, the shape of the cytoplasm appears to be tightened by an "S-" or "C-" shaped flagellum. We propose naming this peculiar morphotype "hyperpachymorph", and adding its description to that of T. congolense forest-type. Thus T. (Nannomonas) forms would include: sphaeromorph or "latent body-form" (globular), hyperleptomorph (rodhaini-form, very long and slender, with a free flagellum); leptomorph (simiae-form, slender, with a free flagellum); isomorph (congolense-form, short, generally without a free flagellum); pachymorph (montgomeryi-form, short and stout; 0.25 < WLr < 0.34, without a free flagellum), and hyperpachymorph ("hyper montgomeryi-form", short and very stout; 0.35 < WLr < 0.7, without a free flagellum).

Natural infection of cattle and tsetse flies in South Africa with two genotypic groups of Trypanosoma congolense.

The polymerase chain reaction was used to detect trypanosomes in samples collected from cattle, wild animals and tsetse flies in KwaZulu-Natal Province, South Africa. A total of 673 samples from cattle and 266 from tsetse flies in the study area located near the Hluhluwe-Umfolozi Game Reserve were analysed. Both Trypanosoma congolense and T. vivax were found as single or mixed infections in cattle and tsetse flies. Moreover, the T. congolense in the infections were found to comprise 2 genotypic groups: the Savannah-type and the Kilifi-type, which were present either as single or mixed infections in cattle and in tsetse flies.

Cross-protection between Trypanosoma congolense strains of low and high virulence.

The aim of this study was to assess the existence of possible cross-protection between Trypanosoma congolense strains of low and extreme virulence circulating in the same trypanosomiasis focus. Groups of six mice were infected using one of three strains of low virulence and challenged with one of three strains of extreme virulence. A group of six mice was used as control for each strain of low and extreme virulence. The results showed that mice infected with one of the strains of extreme virulence developed high parasitaemia and a significant drop of the PCV compared to mice infected with a strain of low virulence and challenged with one of the strains of extreme virulence. With an exception of one strain of extreme virulence (strain F), the survival time of mice infected with the strains of extreme virulence was shorter compared to mice infected with strains of low virulence and subsequently challenged with a strain of extreme virulence. These results suggest that in an area where trypanosomes of various virulence profiles circulate, livestock infected with T. congolense strains of low virulence can be protected against the adverse effects of extremely virulent T. congolense strains.

Genetic diversity of trypanosome species in tsetse flies (Glossina spp.) in Nigeria.

BACKGROUND: Trypanosomes cause disease in humans and livestock in sub-Saharan Africa and rely on tsetse flies as their main insect vector. Nigeria is the most populous country in Africa; however, only limited information about the occurrence and diversity of trypanosomes circulating in the country is available. METHODS: Tsetse flies were collected from five different locations in or adjacent to protected areas, i.e. national parks and game reserves, in Nigeria. Proboscis and gut samples were analysed for trypanosome DNA by molecular amplification of the internal transcribed spacer 1 (ITS1) region and part of the trypanosome specific glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. RESULTS: The most abundant Trypanosoma species found in the tsetse gut was T. grayi, a trypanosome infecting crocodiles. It was ubiquitously distributed throughout the country, accounting for over 90% of all cases involving trypanosomes. Trypanosoma congolense was detected in gut samples from all locations except Cross River National Park, but not in the proboscis, while T. brucei (sensu lato) was not detected at all. In proboscis samples, T. vivax was the most prominent. The sequence diversity of gGAPDH suggests that T. vivax and T. grayi represent genetically diverse species clusters. This implies that they are highly dynamic populations. CONCLUSIONS: The prevalence of animal pathogenic trypanosomes throughout Nigeria emphasises the role of protected areas as reservoirs for livestock trypanosomes. The genetic diversity observed within T. vivax and T. grayi populations might be an indication for changing pathogenicity or host range and the origin and consequences of this diversity has to be further investigated.

Remarkable richness of trypanosomes in tsetse flies (Glossina morsitans morsitans and Glossina pallidipes) from the Gorongosa National Park and Niassa National Reserve of Mozambique revealed by fluorescent fragment length barcoding (FFLB).

Trypanosomes of African wild ungulates transmitted by tsetse flies can cause human and livestock diseases. However, trypanosome diversity in wild tsetse flies remains greatly underestimated. We employed FFLB (fluorescent fragment length barcoding) for surveys of trypanosomes in tsetse flies (3086) from the Gorongosa National Park (GNP) and Niassa National Reserve (NNR) in Mozambique (MZ), identified as Glossina morsitans morsitans (GNP/NNR=77.6%/90.5%) and Glossina pallidipes (22.4%/9.5%). Trypanosomes were microscopically detected in 8.3% of tsetse guts. FFLB of gut samples revealed (GNP/NNR): Trypanosoma congolense of Savannah (27%/63%), Kilifi (16.7%/29.7%) and Forest (1.0%/0.3%) genetic groups; T. simiae Tsavo (36.5%/6.1%); T. simiae (22.2%/17.7%); T. godfreyi (18.2%/7.0%); subgenus Trypanozoon (20.2%/25.7%); T. vivax/T. vivax-like (1.5%/5.2%); T. suis/T. suis-like (9.4%/11.9%). Tsetse proboscises exhibited similar species composition, but most prevalent species were (GNP/NNR): T. simiae (21.9%/28%), T. b. brucei (19.2%/31.7%), and T. vivax/T. vivax-like (19.2%/28.6%). Flies harboring mixtures of trypanosomes were common (~ 64%), and combinations of more than four trypanosomes were especially abundant in the pristine NNR. The non-pathogenic T. theileri was found in 2.5% while FFLB profiles of unknown species were detected in 19% of flies examined. This is the first report on molecular diversity of tsetse flies and their trypanosomes in MZ; all trypanosomes pathogenic for ungulates were detected, but no human pathogens were detected. Overall, two species of tsetse flies harbor 12 species/genotypes of trypanosomes. This notable species richness was likely uncovered because flies were captured in wildlife reserves and surveyed using the method of FFLB able to identify, with high sensitivity and accuracy, known and novel trypanosomes. Our findings importantly improve the knowledge on trypanosome diversity in tsetse flies, revealed the greatest species richness so far reported in tsetse fly of any African country, and indicate the existence of a hidden trypanosome diversity to be discovered in African wildlife protected areas.

Genetic structure of Trypanosoma congolense "forest type" circulating in domestic animals and tsetse flies in the South-West region of Cameroon.

Despite the economic impact of trypanosome infections, few investigations have been undertaken on the population genetics and transmission dynamics of animal trypanosomes. In this study, microsatellite markers were used to investigate the population genetics of Trypanosoma congolense "forest type", with the ultimate goal of understanding its transmission dynamics between tsetse flies and domestic animals. Blood samples were collected from pigs, sheep, goats and dogs in five villages in Fontem, South-West region of Cameroon. In these villages, tsetse were captured, dissected and their mid-guts collected. DNA was extracted from blood and tsetse mid-guts and specific primers were used to identify T. congolense "forest type". All positive samples were genetically characterized with seven microsatellite markers. Genetic analyses were performed on samples showing single infections of T. congolense "forest type". Of the 299 blood samples, 137 (46%) were infected by T. congolense "forest type". About 3% (54/1596) of tsetse fly mid-guts were infected by T. congolense "forest type". Of 182 samples with T. congolense "forest type", 52 were excluded from the genetic analysis. The genetic analysis on the 130 remaining samples revealed polymorphism within and between subpopulations of the target trypanosome. The dendrogram of genetic similarities was subdivided into two clusters and three sub-clusters, indicating one major and several minor genotypes of T. congolense "forest type" in tsetse and domestic animals. The low FSTvalues suggest low genetic differentiation and no sub-structuration within subpopulations. The same T. congolense genotypes appear to circulate in tsetse and domestic animals.

Molecular identification of trypanosome species in trypanotolerant cattle from the south of Gabon.

The aim of this study was to provide information on trypanosome species infecting trypanotolerant cattle from southern Gabon. The study was conducted on 224 trypanotolerant cattle from three regions located in southern Gabon, using ITS1 primer-based PCR. Seventy-two (32%) N'dama cattle were found polymerase chain reaction (PCR) positive with trypanosomes. The overall prevalence of trypanosomosis was 57% (63/110), 4% (4/100), and 36% (5/14) in the Gala section of the Nyanga ranch, the Miyama ranch, and Ossiele, respectively. Trypanosoma congolense and Trypanosoma vivax were identified. In Gala section and Ossiele, T. congolense and T. vivax were found. In the Miyama ranch, only T. vivax was identified. Mixed infections were also found. The forest (9%) and savannah (63%) subgroups of T. congolense were identified. The presence of the two subgroups was detected in 16 out of 56 cattle (29%). T. congolense and T. vivax would appear to be the main agents responsible for bovine trypanosomosis in southern Gabon. Although trypanotolerant, N'dama cattle may serve as a reservoir, and this should be further studied. On the other hand, these trypanotolerant cattle can be reared in such tsetse infested areas, which gives them an advantage compared to other trypanosensitive breeds, and this shows that they represent a key factor in biodiversity which has to be promoted.

Comparison of the virulence of Trypanosoma congolense strains isolated from cattle in a trypanosomiasis endemic area of eastern Zambia.

The virulence of 31 genetically different Trypanosoma congolense strains belonging to the Savannah subgroup and isolated from cattle at 11 sites in a trypanosomiasis endemic area of eastern Zambia was compared. Virulence testing, done in OF1 mice, revealed three virulence categories. Strains were considered extremely virulent when the median survival time ranged between 5 and 9 days. Moderately virulent strains had a median survival time between 10 and 30 days and low virulence, more than 30 days. For each strain, the prepatent period was determined and the PCV of the infected animals was measured at regular intervals. A total of six (19.4%) strains belonged to the extremely virulent category with a short prepatent period (mean 2.3+/-0.3 days), high parasitaemia, decline in PCV of 15.6+/-1.1% during the first 7 days p.i. and a short median survival time (mean 6 days). The remainder of the strains belonged to the moderate (13 strains) or low (12 strains) virulence categories with median survival times of 13 and 60 days, respectively. They had longer prepatent periods (means 3.2+/-1.6 days and 3.5+/-1.6 days for moderately virulent and strains with low virulence, respectively) and the decline in PCV was less steep (decline of 14.2+/-0.6 and 9.7+/-0.6% during the first 7 days of infection with moderately virulent strains and strains with low virulence, respectively). Extremely virulent strains were isolated from cattle at four sampling sites with 60% of the cattle from one sampling site harbouring such extremely virulent strains. Results from this study demonstrated substantial differences in the virulence of T. congolense strains of the Savannah subgroup, isolated in one geographic area from a single host species. On the assumption that information on virulence obtained from tests in mice can be extrapolated to cattle, the high proportion of strains with low to moderate virulence is thought to be attributed to the important role of susceptible cattle as reservoirs of trypanosomes in the study area and the ensuing selection against extremely virulent strains.

A modified AFLP for Trypanosoma congolense isolate characterisation.

The amplified fragment length polymorphism (AFLP) technique is a reliable and powerful DNA fingerprint tool for genetic characterisation and analysis. In this paper, we described a modified AFLP with high resolution for Trypanosoma congolense using one enzyme and agarose or Elchrom gel electrophoresis. Eleven allopatric and fourteen sympatric isolates of T. congolense savannah were used to assess the resolution of the method and its ability to characterise T. congolense isolates. Two enzymes (Eco RI or Bgl II) and corresponding non-selective and selective primers were used to identify the most appropriate combination. Patterns generated by Bgl II enzyme and a single selective primer A, C, G or T produced clear profiles. Each of the four selective primers produced different profiles for all the 25 T. congolense isolates. Due to the reduction in the number of bands, profiles could be analysed using agarose or Elchrom gels. Although comparison of a great number of samples could benefit from software help, this technique did not require flurochrome detection methods. The results of the present study demonstrated that this modified AFLP makes the characterisation of T. congolense easier while maintaining high resolution.

Molecular identification of different trypanosome species and subspecies in tsetse flies of northern Nigeria.

BACKGROUND: Animal African Trypanosomiasis (AAT) is caused by several species of trypanosomes including Trypanosoma congolense, T. vivax, T. godfreyi, T. simiae and T. brucei. Two of the subspecies of T. brucei also cause Human African Trypanosomiasis. Although some of them can be mechanically transmitted by biting flies; these trypanosomes are all transmitted by tsetse flies which are the cyclical vectors of Trypanosoma congolense, T. godfreyi, T. simiae and T. brucei. We present here the first report assessing the prevalence of trypanosomes in tsetse flies in Nigeria using molecular tools. METHODS: 488 tsetse flies of three species, Glossina palpalis palpalis, G. tachinoides and G. morsitans submorsitans were collected from Wuya, Niger State and Yankari National Park, Bauchi State in 2012. Trypanosomes were detected and identified using an ITS1 PCR assay on DNA purified from the 'head plus proboscis' (H + P) and abdomen (ABD) parts of each fly. RESULTS: T. vivax and T. congolense Savannah were the major parasites detected. Trypanosomes prevalence was 7.1 % in G. p. palpalis, 11.9 % in G. tachinoides and 13.5 % in G. m. submorsitans. Prevalences of T. congolense Savannah ranged from 2.5 to 6.7 % and of T. vivax were approximately 4.5 %. Trypanosoma congolense Forest, T. godfreyi and T. simiae were also detected in the site of Yankari. The main biological and ecological determinants of trypanosome prevalence were the fly sex, with more trypanosomes found in females than males, and the site, with T. congolense subspp. being more abundant in Yankari than in Wuya. As expected, the trypanosome species diversity was higher in Yankari National Park than in the more agricultural site of Wuya where vertebrate host species diversity is lower. CONCLUSIONS: Our results show that T. congolense Savannah and T. vivax are the main species of parasite potentially causing AAT in the two study sites and that Yankari National Park is a potential reservoir of trypanosomes both in terms of parasite abundance and species diversity.

Prevalence of pathogenic trypanosomes in anaemic cattle from trypanosomosis challenged areas of Itezhi-tezhi district in central Zambia.

BACKGROUND: The measure of anaemia status using packed cell volume (PCV) can be a reliable indicator of African trypanosomosis (AT) in the absence of other anaemia-causing conditions. However, studies that have estimated prevalence of anaemia in cattle from AT endemic areas have rarely reported the prevalence of the disease in the anaemic cattle. Therefore we investigated the prevalence of AT in anaemic cattle at sites that had recently reported the disease in Itezhi tezhi district of central Zambia. METHODS: During a survey, blood samples were collected from 564 randomly selected cattle for anaemia determination from seven crush pens (Mutenda, Kapulwe, Banachoongo, Itumbi, Iyanda, New Ngoma and Shinampamba). At a PCV- value cut off of 26 %, all samples positive for anaemia were subjected to both parasitological examination on thick and thin blood smears and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for detection of trypanosome DNA. Fisher's exact test and a mixed effect logistic regression analyses were used to determine and measures associations, respectively. RESULTS: Of 564 cattle screened, 58 (10.3 %; 95 % CI: 7.8-12.8 %) had anaemia. PCR-RFLP results showed that 17 (29.3 %; 95 % CI; 17.2-41.4 %) anaemic cattle were positive for pathogenic trypanosomes compared to 1 (1.7 %; 95 % CI: 0.0-5.2 %) on parasitological examination using thick smears. The infections were caused by Trypanosoma congolense and Trypanosoma vivax. Fisher's exact test showed a strong association between PCV and pathogenic trypanosome infection (P = 0.004). A mixed effect multivariate logistic regression showed that a one unit increase in PCV reduced the likelihood of detecting AT with PCR-RFLP by 24.7 % (95 % CI: 4.6-40.6 %; P = 0.019) in anaemic cattle, taking into account their age and sex, with random effects for crush pen. CONCLUSION: These results suggest that T. congolense and T. vivax could be important causes of anaemia in cattle reared in AT endemic areas of Itezhi tezhi in Central Zambia. This also suggests that even though pathogenic trypanosomal infection was strongly associated with PCV, it could only account for up to 41 % of the anaemia in cattle. Therefore further investigation to ascertain other factors responsible for anaemia in AT endemic areas of Itezhi tezhi in Central Zambia is needed.

Trypanosome infection rates in tsetse flies in the "silent" sleeping sickness focus of Bafia in the Centre Region in Cameroon.

BACKGROUND: The Bafia sleeping sickness focus of Cameroon is considered as "silent" with no case reported for about 20 years despite medical surveys performed during the last decades. In this focus, all epidemiological factors that can contribute to trypanosomes transmission are present. To update our knowledge on the current risks of Human and Animal African trypanosomiases, different trypanosome species were identified in midguts of tsetse flies captured in the Bafia focus. METHODS: Tsetse flies were trapped using pyramidal traps. Each tsetse fly was identified and live flies were dissected and their midguts collected. DNA was extracted from each midgut and thereafter, blood meals and different trypanosome species were identified with molecular tools. The biological data were transported onto maps in order to have their distribution. RESULTS: Of the 98 traps set up, 461 Glossina palpalis palpalis were captured; 322 (69.8 %) tsetse flies were dissected and 49 (15.2 %) teneral flies identified. The average apparent density of tsetse flies per day was 1.18. Of the 35 (10.9 %) blood meals collected, 82 % were taken on pigs and 17.6 % on humans. Eighty two (25.5 %) trypanosome infections were identified: 56 (17.4 %) T. congolense savannah, 17 (5.3 %) T. congolense forest, 5 (1.6 %) T. vivax and 4 (1.2 %) T. brucei s.l. No infection of T. simiae and T. b. gambiense was identified. Sixty seven (81.7 %) infections were single and 15 (18.3 %) mixed involving one triple infection (T. congolense forest, T. brucei and T. vivax) and 14 double infections: 11 T. congolense forest and T. congolense savannah, two T. congolense savannah and T. brucei, and one of T. brucei and T. vivax. The generated maps show the distribution of tsetse flies and trypanosome infections across the focus. CONCLUSION: This study has shown that animal trypanosomes remain an important problem in this region. Meanwhile, it is very likely that HAT does not seem anymore to be a public health problem in this focus. The generated maps enabled us to define high risk transmission areas for AAT, and where disease control must be focused in order to improve animal health as well as the quantity of animal proteins.

Shared surface epitopes among trypanosomes of the same serodeme expressing different variable surface glycoprotein genes.

African trypanosomes evade the immune response of the mammalian host by undergoing antigenic variation, caused by sequence changes in a variable surface glycoprotein (VSG). The majority of trypanosome clones analyzed thus far are not known to share surface exposed epitopes or express appreciably homologous VSGs. We show here that four clones of Trypanosoma brucei from the same serodeme express different VSGs and share exposed epitopes to varying degrees, as defined by monoclonal antibodies. Rabbit antiserum against any one of the four VSGs recognizes epitopes present on all four trypanosomes in live cell immunofluorescence assay. The expressed VSGs are partially homologous at the N-terminus with multiple point substitutions of amino acids which distinguish each of the four VSGs. The genes coding for these VSGs are members of one gene family and an expression-linked copy with a unique restriction map is present in each trypanosome. Analysis of the ontogeny of the expressed genes should reveal mechanisms of evolution in trypanosome variable antigen repertoires.

The chromosome profiles of Trypanosoma congolense isolates from Kilifi, Kenya and their relationship to serodeme identity.

Chromosomal DNA from 117 Trypanosoma congolense clones from 54 stocks, isolated from cattle introduced onto a ranch in Kilifi in the coastal area of Kenya, was fractionated by the orthogonal field alternation gel electrophoresis technique. The technique resolved chromosomes in the size range of 100 kb-1 Mb. The chromosome profile for cloned trypanosome populations was relatively stable with regard to number and size of the chromosome bands following transmission in mice, cattle, goats or tsetse flies. Only in one clone was a shift observed in the position of one medium-sized chromosome band following cyclical development in tsetse. On the basis of their chromosome profiles, the 117 clones could be divided into 18 distinct groups. Representative clones, randomly selected from 7 of the 18 chromosome profile groups were inoculated into steers and goats in order to raise variable antigen type (VAT) repertoire-specific infection sera. Cross-neutralization assays demonstrated that recovery sera from animals infected with a clone neutralized all the clones with an identical chromosome profile. This suggests that clones having an identical chromosome profile also express an identical VAT-repertoire (serodeme).

Comparative structural analysis of calmodulins from Trypanosoma brucei, T. congolense, T. vivax, Tetrahymena thermophila and bovine brain.

Calmodulin is an intracellular calcium receptor protein utilized extensively by eukaryotic cells to mediate responsiveness to calcium signals. The present study evaluates the effects on protein structure of amino acid substitutions in trypanosome calmodulin. Calmodulin conformation, hydrophobicity and antigenic determinants are compared among Trypanosoma brucei, Trypanosoma congolense, Trypanosoma vivax, Tetrahymena thermophila and bovine brain. Trypanosome calmodulin differs from brain and Tetrahymena calmodulins based upon isoelectric point, retention time on a C-2/C-18 reverse phase column and interaction with polyclonal antibodies against trypanosome calmodulin by radioimmunoassay or Western procedures. These same analyses do not distinguish trypanosome calmodulins from each other. Polyclonal antibodies against Tetrahymena calmodulin are equally specific and do not recognize the trypanosome or brain calmodulins. Calcium-induced exposure of hydrophobic binding sites are quantitated using the fluorescent probe, N-phenyl-1-naphthylamine. All calmodulins, regardless of source, enhance the fluorescence of N-phenyl-1-naphthylamine 3-4 fold in the presence of calcium. These data demonstrate the extent to which functional calmodulins vary in their structures. We conclude that African trypanosomes share a common calmodulin that is structurally distinct from calmodulin of vertebrates or Tetrahymena.

DNA content and molecular karyotype of trypanosomes of the subgenus Nannomonas.

The relative DNA contents of representative stocks of 5 groups within the trypanosome subgenus Nannomonas (Trypanosoma simiae, Godfreyi, T. congolense Savannah, Forest and Kilifi) were measured by flow cytometry. The range of DNA contents formed a continuum. Nevertheless small differences were observed between the groups, with T. simiae/T. congolense Savannah and T. congolense Kilifi/Forest at the lower and higher ends of the range respectively. Analysis of karyotype by pulsed field gel electrophoresis showed all the 5 Nannomonas groups to have minichromosomes and variable numbers of small chromosomes in the 100-700 kb size range. The size and relative number of mini-chromosomes varied from group to group, but no correlation between molecular karyotype and DNA content was observed.

A preliminary comparison of Trypanosoma simiae and T. congolense by isoenzyme electrophoresis.

Trypanosoma simiae, although similar to T. congolense in morphology and mode of development in the tsetse vector, is regarded as a separate species mainly because it is highly pathogenic to the domestic pig and fails to infect rodents. To establish whether the two species are distinct biochemically, we compared by isoenzyme electrophoresis 2 isolates of T. simiae with 7 stocks of T. congolense, together with one of T. brucei. All isoenzyme patterns of the 6 enzymes examined differed in T. simiae from T. congolense and T. brucei stocks. This supports the designation of T. simiae as a separate species. However, a comparison involving a much larger collection of T. simiae is necessary in order to get conclusive evidence.

Drug sensitivity screening in vitro of populations of Trypanosoma congolense originating from cattle and tsetse flies at Nguruman, Kenya.

An in vitro assay that utilises in vitro-derived metacyclic trypanosomes was used to determine the drug sensitivity of 7 populations of Trypanosoma congolense collected from cattle and tsetse flies at Nguruman; a trypanosomiasis-endemic area in southwest Kenya. The metacyclic trypanosomes used in the assay were obtained from cultures initiated directly from either the blood of cattle with low levels of parasitaemia or from guts of infected tsetse flies. Sensitivities to isometamidium chloride, diminazene aceturate and homidium salts were assessed at various drug concentrations (0.5 ng-50 micrograms/ml). The results were compared with those obtained with two characterised laboratory populations. In spite of the fact that isometamidium chloride had not been widely used at Nguruman, two of the stocks (KE 3302 and KE 3303) expressed high levels of resistance to this drug (resistant to 100 ng/ml and 1000 ng/ml, respectively). In contrast, all of the populations examined were as sensitive, or more so, to diminazene aceturate than the sensitive laboratory clone IL 1180; two stocks (KE 3305 and KE 3306) were more sensitive (resistant to 0.5 microgram/ml, sensitive to 1 microgram/ml) and three stocks expressed the same level of sensitivity as IL 1180 (resistant to 1 microgram/ml, sensitive to 5 micrograms/ml). Since the results of the in vitro assay correlated well with field observations it was concluded that the assay would be a useful tool in epidemiological studies to determine the resistance phenotypes of trypanosome populations in the field, thereby enabling development of appropriate control measures for particular areas.

Identification of trypanosomes isolated by KIVI from wild mammals in Côte d'Ivoire: diagnostic, taxonomic and epidemiological considerations.

In Côte d'Ivoire, a comparative study was carried out on 122 wild mammals by parasitological and serological examination and by in vitro isolation of trypanosomes from fresh blood (KIVI). Thirteen isolated stocks were studied by isoenzymes and compared with Trypanosoma congolense and T. brucei bouaflé group reference stocks. Of the 122 animals, only 22 were positive on blood smears while 88 were KIVI positive and 92 were CATT/T. b. gambiense positive. For six stocks identified by isoenzymes as T. congolense, the agreement between ELISA and CATT was good (75%). As compared with CATT, antigen detection ELISA was not satisfactory for T. brucei (20%). Out of 18, 16 stocks represented a separate zymodeme (seven T. congolense and nine T. brucei) and a high genetic heterogeneity was observed. For T. congolense, savanna, kilifi and forest groups were represented by one zymodeme each. The four remaining zymodemes while put into this T. congolense group, were strongly independent of each other. Morphology indicated that those new zymodemes correspond to T. congolense. In the other hand, five new zymodemes fit into T. brucei classification.

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.