Trypanosoma congolense pdf

Need an account? Click here to sign up. Download Free PDF. Trypanosoma congolense: the distribution of enzymic variants in East and West Africa Parasitology, Rose Baker. A short summary of this paper. Trypanosoma congolense: the distribution of enzymic variants in East and West Africa. Parasitology The zymodemes were grouped, both from a dendrogram and using a cladistic method, after calculating the dissimilarity, or distance, between profiles.

Previous obser- vations are broadly confirmed, the zymodemes clustering separately according to geographi- cal origin and ecological zone. Thus, one group was composed almost entirely of East African stocks, and another of stocks from both East and West Africa, although each group was of savanna origin. A third group was composed of stocks from the humid, rain-forest zones of West Africa, and was particularly characterized by isoenzyme variants of superoxide dismutase and glucose-phosphate isomerase.

Two stocks from the Kenyan coast formed a markedly separate group, which may be taxonomically distinct. However, its epidemiology is still poorly understood, especially regarding the distribution of the different strains.

Several, at least, are known to differ in such attributes as the clinical disease they cause, infectivity and distribution see Hoare, ; Stephen, Recently, the genetic diversity within T. Thus they had a group consisting of stocks originating from the humid regions of West Africa, and another group of stocks originating from the savanna zone of Africa.

The investigation described in this paper further analyses the relationships between enzymic variants of T. Since the previous study dealt mainly with stocks from West Africa, this investigation concentrates more on stocks from East Africa, especially those isolated from tsetse, but 35 stocks from the previous study are included for comparative purposes. Most were recently isolated in mice by inoculating with organisms from either tsetse flies or domestic animals in Kenya.

When the newly isolated stocks reached a sufficient parasitaemia in the mice, they were stabilated in liquid nitrogen. A total of 28 stocks of West African origin were included for comparison. After comparing 10 enzymes, 6 were chosen for the study as giving the most consistently clear banding.

In this way, maximum information was derived from very small amounts of material. The 6 enzymes were: EC 1. SOD was run for h at V in M phosphate tank buffer, pH , and a gel buffer made up of tank buffer in distilled water.

Diagrammatic representation of the variation in Trypanosoma congolense by iso- enzyme electrophoresis. The lettered boxes on the left-hand side show the coding for the individual bands. The numbers indicate isoenzyme pattern. The striped bands denote weak staining. Photography was under ordinary bright light. In describing the results the following terms are used.

After appropriate staining, a 'band' is defined as a distinct area of enzyme activity on a gel, and is identified by its position relative to a standard marker band. Bands are numbered alphabetically with small letters progressing from the cathode end.

The 'pattern' is a unique arrangement of bands for a particular enzyme in an organism. The different patterns of the same enzyme in separate organisms are numbered with arabic numerals for ease of computer entry; these numbers only indicate the chronological sequences of discovery.

A 'profile' comprises the isoenzyme patterns for every enzyme used to characterize a stock. A 'zymodeme' is a collection of stocks with identical enzyme profiles. The patterns seen in the stocks for the 6 enzymes compared are in Fig. A total of 71 different profiles was recorded. The method used for measuring relationships between profiles was calculation of the Euclidean distance Ward, , where the distance between two OTUs is the sum of the squared character differences.

For bands coded as 1 or 0 for presence or absence, this is equivalent to taking the distance between two profiles as the number of non-matching bands. From these calculations, distance matrices for the OTUs were constructed. Some isoenzyme bands may represent heterozygous products and may therefore be used in estimating allelic frequencies of genotypes for the purpose of studying the genetic composition of a population. However, this is not taken into account here and will appear in a separate paper.

For comparison with earlier work, two approaches were used. In the first, the more conventional Ward's method Ward, was used to construct a dendrogram from the profiles. In this technique, profiles are progressively grouped together until one group remains. At each step, two groups of profiles are clustered together. Ward's method uses a simple criterion based on the distance matrix to decide which two groups should be clustered at any stage. In the second approach, a cladogram was constructed from the profiles.

This is an evolutionary or phylogenetic tree, and the dissimilarity between two profiles is the distance between them through the cladogram, the 'patristic' distance.

The cladogram was then progressively broken down into the most likely clusters. The method was iterative; as the bands were weighted, those changing least often were assumed to have had the greatest influence in the course of evolution and were therefore weighted the most. In addition, the expected stage-specific cell surface markers were expressed by each of three life cycle stages procyclics, epimastigotes and metacyclics cultured in vitro [ 32 ].

The recent publication of the genome sequence of T. This has been dealt with rather cursorily in the literature, probably because of its similarity to that of T. A key question is whether there is a form equivalent to the asymmetric divider of T. Here we have examined a detailed timecourse of the development of T. Experimental tsetse flies were from the Bristol laboratory colony of Glossina morsitans morsitans originally from Zimbabwe.

Male and female flies were used for experiments, being given the infective bloodmeal for their first feed 24—48 hours post-eclosion.

For examination of trypanosomes extruded in spit samples a mixture of saliva and regurgitated foregut contents , flies were caged individually; for other experiments, flies were caged in groups of 15— Bloodstream form trypanosomes of T.

Alimentary tracts, from the proventriculus to the rectum, were dissected in a drop of phosphate buffered saline PBS and viewed as wet mounts under phase contrast x magnification to search for trypanosomes. Proventriculi were removed from infected midguts and viewed separately. Proboscides from flies with midgut infection were dissected into a separate drop of PBS and teased apart, gently rubbing a fine needle down the length of the proboscis to dislodge the trypanosomes.

Cibaria from flies with midgut infection were dissected into a separate drop of PBS. Bloodstream form trypanosomes obtained from a mouse infected with T. Spit samples were obtained from individually caged flies essentially as described by [ 41 ].

Saliva samples dried immediately on contact with the microscope slide and slides were stored in the dark at ambient temperature before examination. Each image was photographed under phase contrast and UV fluorescence at x magnification.

Measurements were made on the digital images using Image J software Version 1. Dimensions measured were those used by [ 11 ] and shown in Additional file 1 : Figure S1. Only three uncorrelated factors were identified with eigenvalues greater than 1, accounting for Extracted scores for factors 1 and 2 for each trypanosome were plotted.

Loadings were extracted and the absolute values plotted Additional file 2 : Figure S2 to determine the extent to which each of the individual measurements contributed to the first two factors. See legend to Additional file 2 : Figure S2 for details. A high proportion of G. Flies were more susceptible to infection with this trypanosome strain than with T.

In contrast, many T. Bloodstream form BSF T. These changes reiterate those observed for differentiation of T. Morphology of developmental stages of Trypanosoma congolense. Bloodstream forms. Midgut procyclic trypanosomes gradually lengthened over time, from a mean length of At the same time, the trypanosomes became more slender, halving in width from 2. Corresponding changes were seen in nuclear length and width, the nucleus becoming longer and thinner Figures 1B to E ; Additional file 3 : Table S1.

Keeping the kinetoplast as the point of reference, most of the increase in cell length appeared to occur at the anterior end of the cell: the kinetoplast-anterior distance increased from The relative distance between the nucleus and kinetoplast remained constant Additional file 3 : Table S1.

Assuming that the increase in cell length results from posterior extension of microtubules as in T. It is important to note that at any one timepoint the trypanosome population was not of uniform morphology, but showed large variability both in length and shape Additional file 3 : Table S1. For example, in some trypanosomes the posterior tapered to a point, while in others the posterior was blunt.

Cell division was symmetrical, yielding two trypanosomes of similar size Figure 1J. Trypanosomes appeared in the proventriculus as early as six days after infection. Only trypomastigotes were observed Figure 2A , although a few cells with the kinetoplast posterior but very close to the nucleus were also found; the juxtaposition of the kinetoplast and nucleus in these cells produced a marked widening or bulge Figure 2B , reminiscent of a cell type found in the proboscis in T.

Proventricular trypanosomes. Trypomastigote with posterior kinetoplast. Trypomastigotes with the kinetoplast adjacent and to the posterior pole of the nucleus; the cell is distended in the region of the kinetoplast and nucleus. Principal components analysis PCA of the morphological parameters of the proventricular trypanosomes over time shows that they constitute a very discrete population, particularly early on in the infection time course days 9—10, Figure 3A. Strikingly, although PCA identifies latent variables, which are uncorrelated over the whole population, there was a strong correlation between factors 1 and 2 in the proventricular trypanosomes at day 9 Figure 3A , top panel.

This reflects the fact that this population was unusually uniform in terms of the relationship between cell length and relative organelle positioning with respect to the anterior and posterior poles of the cell. The variables that contributed most to PCA factor 1 were cell length, nuclear length and the distances of the kinetoplast and nucleus from the anterior end of the cell, while for factor 2 the key variables were the distance between the kinetoplast and nucleus and the distances of the kinetoplast and nucleus from the posterior end of the cell Additional file 2 : Figure S2.

Principal components analysis PCA. Each plot shows scores for PCA factor 1 versus factor 2 derived from the mensural data from individual trypanosomes, each represented by a coloured dot. Sequential plots of proventricular trypanosomes black dots compared with all other trypanosomes grey dots. Comparison of bloodstream forms red dots , midgut, proboscis and cibarium trypomastigotes grey dots , epimastigotes green dots , and metacyclics blue dots.

Comparison of trypanosomes from the proboscis and cibarium. The spit is a mixture of saliva and regurgitated foregut contents, but only foregut trypanosomes are represented before a proboscis infection is established.

Spit from two flies was already trypanosome-positive on day 10 and the number of positive flies steadily increased during the timecourse of infection Figure 4. By day 23, 34 flies had produced at least one trypanosome-positive spit sample. On dissection, it was found that only 42 of the 50 individually-caged flies had an infected midgut and hence were capable of producing a trypanosome-positive spit sample; 39 flies had an infected proboscis Table 1.

Trypanosoma congolense in spit samples. Cumulative percentage of infected flies that produced a trypanosome-positive spit sample. Initially only trypomastigotes were present in the spit samples, until epimastigotes appeared on day 15 Figure 5A , B, C.

The trypomastigotes were not noticeably different from those found in the proventriculus, except for a few very long forms found on day 16 Additional file 4 : Table S2 , suggesting that the migratory trypanosomes in the foregut are simply proventricular trypanosomes that have passed through the peritrophic matrix into the foregut lumen.

This contrasts with T. Mixture of trypomastigotes with epimastigote arrowed. Epimastigote with long posterior arrowed. Epimastigote 2K1N. Metacyclics from day 21; contrast the size of metacyclics with the other trypomastigotes from the proboscis shown in panel A at the same scale. This accords with previous findings that the epimastigotes arise in the proboscis, not the proventriculus or foregut [ 21 , 34 ]. The morphology of epimastigotes in the spit and proboscis was similar.

The posterior was sometimes extremely elongated, such that the nucleus was positioned equidistant between the posterior and anterior poles of the cell Figure 5A and B ; the elongated posterior sometimes twisted during fixation Figure 5B and C. Whether the nozzle phenotype is analogous in T. The close proximity of the kinetoplast to the nucleus in the epimastigotes was associated with a bulge in the cell near the nucleus, particularly in dividing cells Figures 5A-C ; this was also observed for some proventricular trypomastigotes where the kinetoplast and nucleus were juxtaposed, but in that case the kinetoplast was posterior not anterior to the nucleus Figure 3B.

Metacyclics first appeared in spit samples on day 21, identified by their very short length In metacyclics, the kinetoplast was very close to the posterior pole of the cell Figure 5D ; Additional file 4 : Table S2. The trypanosome population of the proboscis was highly variable in composition and morphology. Shorter trypomastigotes were also present Figure 6 ; these were sometimes observed in division Figure 6B , C , suggesting they are pre-metacyclics.

Few metacyclics were recovered from proboscides at dissection and were morphologically similar to those observed in spit samples Figure 5D ; Additional file 5 : Table S3.

The length of epimastigotes increased with duration of infection, with some extremely long cells present from day 19 onwards Figure 7A ; the average length showed a marked increase, almost doubling between days 13 and 19 Additional file 5 : Table S3. This echoes the observations from in vitro studies where epimastigotes are reported to contract and shorten soon after attachment to the plastic substratum and then to lengthen after a few days [ 26 , 30 , 31 ].

In live trypanosomes the elongated posterior had a rigid appearance, contrasting with the fluid undulating motion of trypomastigotes Additional file 6 : Movie 1. As in the epimastigotes from spit samples, the elongated posterior was often twisted or crumpled during fixation, and sometimes had a transparent appearance Figure 7B , C, E. In some trypanosomes the posterior appeared to be truncated, almost looking as if the posterior was broken off or twisted back on itself Figure 7D.

Such trypanosomes were also observed in vivo Additional file 7 : Movie 2 , so this is not an artefact of fixation. Proboscis epimastigotes. Epimastigote with long posterior broad arrow ; the kinetoplast arrow is adjacent and anterior to the nucleus. Epimastigotes with transparent posterior extensions broad arrows ; position of kinetoplast is indicated by thin arrow; in C the posterior extension is crumpled; D shows a truncated form.

The cibarium is a widening of the alimentary tract that lies between the proboscis and foregut; the chitin-lined walls act as a pump allowing the fly to suck blood [ 47 ]. Both T. Trypanosomes were found widely distributed across the dorsal wall as described [ 18 ] Additional file 8 : Movie 3. The morphology of these cells was similar to that of trypomastigotes and epimastigotes in the proboscis, suggesting that the cibarium harbours an extension of the proboscis population rather than a separate morphological stage.

This is clearly seen in the PCA comparing trypanosomes from the cibarium and proboscis; there is considerable overlap between these two groups Figure 2. The transition from trypomastigote to epimastigote involves the re-positioning of the kinetoplast relative to the nucleus. First the kinetoplast-nuclear distance diminishes before the two organelles pass by each other until the kinetoplast is fully anterior; the kinetoplast-nuclear distance then increases.

As this is a gradual process, the point of transition from trypomastigote to epimastigote is uncertain. Comparison of proboscis trypanosomes at these various stages of transition shows that the longest cells are those with the kinetoplast fully anterior to the nucleus Table 3. The increase in length is due almost entirely to growth of the posterior end of the cell, as shown by comparison of kinetoplast position relative to the posterior or anterior of the cell Table 3. This contrasts with the transition from trypomastigote to epimastigote in T.

The length of the asymmetric divider during this transition phase was fairly constant [ 11 ] and much less variable than that found here for T. Figures 8A-C show examples of epimastigotes apparently giving rise to daughter epimastigotes; in some cases the daughter epimastigote clearly had a very long posterior Figures 8A , suggesting that division may be asymmetric, though the length of the posterior of the parental cell is hard to judge.

Figures 8D, F and G show examples of trypomastigotes apparently giving rise to daughter epimastigotes with posterior ends of modest length; we assume these dividing stages show the transition from proventricular trypomastigotes to epimastigotes that will subsequently attach to the lining of the proboscis, but are cautious of constructing a narrative from a few fixed cells. In contrast, Figure 8E shows an example of the reverse, an epimastigote giving rise to a daughter trypomastigote; this can be interpreted as the first step on the pathway to metacyclic, again with the caveat that these were rarely observed, fixed cells.

The same caveat applies to the curious asymmetric pairs of cells, which also appear to show epimastigotes giving rise to daughter trypomastigotes, but in these examples, very long epimastigotes and very short trypomastigotes Figures 8H and 8I.

The scarcity of dividing stages, coupled with the fact that we sampled only unattached trypanosomes spilt from the proboscis, means that we are unable to state categorically that the transition from trypomastigote to epimastigote, and subsequently from epimastigote to trypomastigote, is always associated with cell division. Proboscis dividing trypanosome.

Fixed and DAPI-stained trypanosomes in division 2K1N or 2K2N from dissected proboscides; these trypanosomes were free rather than attached inside the proboscis. Panels A-C show examples of epimastigotes apparently giving rise to daughter epimastigotes.

Panels D, F and G show examples of trypomastigotes apparently giving rise to daughter epimastigotes. Since forms Roberts et al It is, therefore, possible the mean plasma concentration of diminazene that trypanosomes would be present in this over the first 48 hours following treatment was extravascular site 24 hours after tsetse have fed 1.

Trypano- double treatment with diminazene aceturate to somes which are morphologically similar to lesion eliminate infections with m in seven out of forms of T congolense have been observed in 10 animals was not surprising. It would therefore vitro Gray et al However, in vitro, this appear that when animals are infected with try- life-cycle stage does not appear to differ from panosomes that express a high level of resistance metacyclic forms in its sensitivity to diminazene to diminazene, double treatment with diminazene M.

Gray, unpublished data. These Aliu et al have described the pharmacoki- animals were infected with IL via the bites netics of diminazene in the blood of goats but of infected tsetse and treated 24 hours later with no data exist for the dermis. It is possible that diminazene aceturate at a dose of mg kg 1 significantlyhigher concentrations ofdiminazene bodyweight. However, in contrast to animals in are attained in the dermis than in blood and this group A, trypanosomes were not detected in any possibility is currently under investigation.

There Such a phenomenon has been described in mice is, therefore, an apparent difference in sensitivity infected with Tbrucei Jennings et al , The basis infections if the drug was administered seven days of this phenomenon is unclear. However, trypanosomes reap- The development of local skin reactions, or peared following administration of the drug if chancres, at the site of inoculation of metacyclic treatment was delayed until 14 days after infection.

While T brucei has been following tsetse transmission of T congolense demonstrated to occur within the CNS of cattle Emery and Moloo and was produced in Masake et al and T vivax has been detected the study described here by both T congolense in the aqueous humour of goats Whitelaw et al IL and Tcongolense IL Peregrine within the CNS of ruminants when animals were this work from the Overseas Development infected simultaneously with both T congolense Administration of the United Kingdom.

This is and T brucei Masake et al ILRAD publication number One further possible explanation for the appar- ent difference in sensitivity of IL between References day 1 and day 19 of infection is that within this ALIU, Y. Journal of Chromatography , the trypanosomes had altered. It is pos- in veterinary medicine. Journal of Veterinary Pharmacology and sible that the same holds true for diminazene. Annals lymph node draining the site of tsetse bites.

The same was true for the reaction A. In In vitro Methods for Parasite before the detection of trypanosomes in the Cultivation. Eds A. Taylor and J. London, Academic blood. Furthermore, enlargement of the draining Press.

In Perspectivesin Trypanosomiasis Research. This is Ed J. Chichester, Research Studio Press. However, unlike Trypanosoma brucei in mice and the efficiency of chemotherapy.

Veterinary Record80, the lymph node draining the site of tsetse bites but were not subsequently detected parasitaemic. McOdimba, Mr A. A cta Adema, Mr S. Scott for his help Berenil diminazene aceturate -resistant Trypanosoma congolense in cattle under natural tsetse challenge at Kibaha, Tanzania.

Acta with the analysis of the data, Ms P. Otieno and Tropica 45, Ms D. Lewa for secretarial assistance and Dr A. An improved parasitological technique for diagnosis of African trypanosomiasis. Parasitology , Extravascular foci of Trypanosoma vivax in goats: the central nervous system and aqueous humor of the eye as potential sources of relapse PINDER, M.