The use of culture-derived metacyclic trypanosomes in studies on the serological relationships of stocks of Trypanosoma brucei gambiense

Metacyclic trypanosomes of five stocks of Trypanosoma brucei gambiense were produced in vitro in tsetse head-salivary gland explant cultures and used to infect rabbits. Sera were collected from the rabbits and monitored by agglutination tests for antibody production to nine serotype antigens of T. b. gambiense. In the case of a Nigerian stock of T. b. gambiense the sequences of antibody production were found to be similar in animals infected with the stock transmitted by tsetse flies and from culture. Many similarities were also found between the patterns of antibody production in rabbits infected with stocks of T. b. gambiense from Senegal, Nigeria, Zaire and Uganda. The occurrence of similar serotypes in geographically different stocks of T. b. gambiense provides further support for continuing efforts to develop improved serodiagnostic tests for sleeping sickness based on variable trypanosome antigens and to find techniques for immunoprophylaxis.     

Effects of immunosuppressive agents on the course of Trypanosoma (trypanozoon) brucei infections in heat-stressed mice

Experimental evidence has been obtained showing that x-irradiation or cyclophosphamide treatment of mice previous to inoculation, elevated the level of parasitaemias in mice infected with T. (T.) brucei when the immunosuppressed animals were maintained at 35C. The trypanosomes in these mice appeared to be less pathogenic than in the control animals kept at (22–27°C) normal room temperature. Many bizarre forms of trypanosomes appeared when the immunosuppressed animals were maintained at the high ambient temperature. It is suggested that the reduced pathogenic effects of these trypanosomes to mice and the occurrence of the various morphological forms are due to direct temperature effect on trypanosome organisms.     

大鼠肾细胞分离制备及培养方法的比较

目的比较胶原酶消化法、胰蛋白酶消化法和胶原酶、胰蛋白酶两步消化法(简称两步消化法)对大鼠肾细胞分离效果及细胞产量的影响,以及不同培养基(MEM、RPMI1640)、鼠龄(乳鼠、成年鼠)对细胞产量、质量、增殖率和乳酸脱氢酶(LDH)漏出量的影响。方法分别对两组Wistar大鼠(乳鼠和成年鼠)用三种不同的消化方法制备大鼠肾组织细胞悬液,分别在MEM培养液和RPMI1640培养液中原代培养,培养48小时之后第一次传代,观察细胞质量,计算细胞产量,MTT法检测传代细胞24~48小时之间的增殖率,生化分析仪检测传代细胞第1~7天培养液中LDH漏出量。结果胶原酶消化法和两步消化法的肾细胞产量显著高于胰蛋白酶消化法(P<0.01),两步消化法的细胞质量优于胶原酶消化法,MEM培养液和RPMI1640培养液对细胞产量、增殖率和LDH漏出量的影响均无显著性差异,乳鼠的肾细胞产量显著高于成年鼠(P<0.01)。结论两步消化法对于肾细胞的分离制备尤为适合,MEM培养液和RPMI1640培养液均适于肾细胞的培养,乳鼠比成年鼠更适合肾细胞的制备。     

Growth characteristics of canine pathogenic viruses in MDCK cells cultured in RPMI 1640 medium without animal protein

Madin Darby canine kidney (MDCK) cells were adapted to serum-free RPMI 1640 medium and used for cultivation of canine viruses. RPMI 1640 medium was supplemented with a soybean peptone, L-glutamine and antibiotics, so that the protein concentration was less than 5 microg/ml (RPMI/SP medium). The resulting adapted MDCK-SP cells showed steady growth after the twenty-eighth passage in RPMI/SP medium (MDCK-SP cell culture). Canine distemper virus, canine parvovirus, canine adenoviruses and canine parainfluenza virus, which are the principal components of canine combined virus vaccines, grew in the MDCK-SP cell culture as efficiently as the parental MDCK cells cultured in the conventional Eagle's MEM containing fetal bovine serum. Consequently, the use of MDCK-SP cell culture can make current canine vaccine products much safer, of higher quality and at lower cost.     

Studies on serum requirements for the cultivation of Plasmodium falciparum. 2. Medium enrichment

Previous experiments using RPMI 1640 medium have indicated that the dialysis of human serum removes components of low relative molecular mass (6000-8000 RMM) that are essential for continuous cultivation of Plasmodium falciparum. To determine which low-RMM components are important for parasite development, we compared growth in normal serum to that in dialysed serum using a number of other commercially available media, which we considered to be richer than RPMI 1640. Through these comparisons, we determined that hypoxanthine was the major dialysable nutrient required for parasite development. High quality bovine serum requires 3 - 12 × 10^-5 mol/litre of hypoxanthine as a supplement to support continuous cultures of P. falciparum. Thus far we have been unable to attain parasite growth in medium containing supplemented bovine serum that is as good as growth in medium containing human serum.     

Physiological relationships and circadian periodicities in rodent trypanosomes

Trypanosome circadian rhythms in rats infected with Trypanosoma lewisi and mice infected with T. duttoni (=T. musculi) were observed. Peak numbers of trypanosomes were recorded at nightfall and fewest organisms seen at daybreak. Reversal of the photoperiod resulted in a comparable reversal of the trypanosome parasitaemia.Periodicities of blood glucose levels and circulating leucocytes were relatively similar in T. lewisi-infected rats to rhythms previously defined in normal animals. In trypanosome-infected mice, circulating leucocytes had a peak at 1800 hours and were minimal at midnight. Immune serum and epinephrine apparently had opposite effects on numbers of circulating rat trypanosomes; antisera reduced and epinephrine increased the numbers. Increased motor activity appeared to induce increased parasitaemia.Results of these and other studies suggest that in diurnally active hosts, trypanosome periodicities are characterized by increasing numbers in the circulation throughout the day and reach a peak just before darkness. In nocturnally active animals a similar rhythm was observed with only a slight phase change; circulating trypanosomes increased throughout the day and were maximal soon after nocturnal onset.     

The use of specific and generic primers to identify trypanosome infections of wild tsetse flies in Tanzania by PCR

The accurate identification of trypanosome species and subspecies remains a challenging task in the epidemiology of human and animal trypanosomiasis in tropical Africa. Currently, there are specific PCR tests to identify about 10 different species, subspecies or subgroups of African tsetse-transmitted trypanosomes. These PCR tests have been used here to identify trypanosomes in four species of tsetse (Glossina brevipalpis, G. pallidipes, G. swynnertoni, G. morsitans morsitans) from two areas of Tanzania. PCR using species-specific primers was performed on 1041 dissection-positive proboscides, giving an overall positive identification in 254 (24%). Of these, 61 proboscides (24%) contained two or more trypanosomes. The trypanosome with the greatest overall prevalence at both field sites was Trypanosoma simiae Tsavo, which was identified in a total of 118 infected tsetse proboscides (46%). At Pangani, T. godfreyi was found in G. pallidipes but not in G. brevipalpis, suggesting that these flies might have different susceptibility to this trypanosome or might have fed on a different range of hosts. A high proportion (about 75%) of trypanosome infections remained unidentified. To investigate the identity of these unidentified samples, we used primers complementary to the conserved regions of trypanosomal small subunit ribosomal RNA (ssu rRNA) genes to amplify variable segments of the gene. Amplified DNA fragments were cloned, sequenced and compared with ssu rRNA genes on database of known trypanosome species. In this way, we have tentatively identified two new trypanosomes: a trypanosome related to Trypanosoma vivax and a trypanosome related to T. godfreyi. The T. godfreyi-related trypanosome occurred frequently in the Tanzanian field samples and appears to be widespread. Molecular identification of these two new trypanosomes should now facilitate their isolation and full biological characterisation.     

Trypanosomiasis in domestic livestock in the Lambwe Valley area and a field evaluation of various diagnostic techniques

A preliminary survey of 2 073 domestic animals in the Lambwe Valley, Kenya, showed a 7.4% rate of infection with Trypanosoma congolense and T. vivax. In comprehensive surveys covering 6 384 domestic stock, pathogenic trypanosomes were found in 17.0% of cattle, 5.0% of sheep, and 2.1% of goats. Adults were more often infected than young animals, and males more often than females. T. congolense was the trypanosome most frequently diagnosed, followed by T. vivax and the T. brucei subgroup. T. theileri was also found. The examination of wet blood films in the field as a means of diagnosing trypanosome infections was shown to be valueless. More infections were detected in peripheral blood films than in systemic blood films, but both should be examined. An examination of smears of glandular fluid is essential for the diagnosis of T. vivax in cattle, while mouse-inoculation tests are necessary for the diagnosis of the T. brucei subgroup. The detection of T. vivax was improved by the high-speed centrifugation of blood samples in capillary tubes.     

Growth of protozoa in tissue culture. III. Trypanosoma cruzi

The technique used for cultivation of exoerythrocytic forms of P. gallinaceum has been applied to the growth of Trypanosoma cruzi also. A suspension of trypanosomes from the blood of an infected mouse was added to cultures of rat embryo. During the first 2 days great numbers of trypanosomes were present in the culture fluid, not particularly associated with the cells. Then they gradually became fewer until by about the 12th day they were rare or absent. Later small numbers reappeared in the culture fluid and persisted there until the end of the culture. Stained preparations made after. 6 days or more showed great numbers of intracellular parasites in all stages between the rounded leishmanoid form and the mature trypanosome form. The parasite occurred in cardiac muscle fibres, in macrophages and in elongated cells with processes (probably reticulo-endothelial cells). Cultures have been maintained for 59 days. As described by Meyer (1942) T. cruzi could also be grown in cultures from the brain of chick embryo.This technique offers favourable conditions for study of the intracellular development of T. cruzi and of the action of drugs upon it.     

A statistical study of the sensitivity of the haematocrit centrifuge technique in the detection of trypanosomes in blood

The sensitivity of the haematocrit centrifuge technique in detecting trypanosomes was investigated using both old laboratory and freshly isolated strains.Two alternative methods of analysing the results are suggested, based on the binomial and Poisson distributions.The analysis shows that the H.C.T. can detect about 85% of the trypanosomes present in capillary samples of Trypanosoma brucei, T. rhodesiense and T. evansii, and 30% of T. congolense group trypanosomes. The technique is therefore much more sensitive than previous wet preparation or thick smear techniques.Graphs are provided showing the minimum number of tubes that would have to be examined for 95% and 99% detection of low infections (i.e., in the range up to 500 trypanosomes per ml.).Analysis of the results using the Poisson model includes details of a simple method that estimates the efficiency of the H.C.T. from the slope of a graph.The present results do not show any significant relationship between the probability that a trypanosome will be missed by the H.C.T. and trypanosome density.     

A comparison of skin-test responses using antigen from Leishmania donovani and a lizard trypanosome

261 individuals were skin tested with leishmanin from Leishmania donovani and with an antigen from a lizard trypanosome isolated in an area endemic for kala-azar in Ethiopia. 83 of the 261 responded positively, with indurations of 5 mm or greater, to one antigen but negatively to the other, and 42 reacted positively to both.Among the Afar people of the Awash valley in north-eastern Ethiopia and the Nyangatom people of south-western Ethiopia, positive response to both antigens was high, though many individuals responded to one antigen but not the other. Both these groups live as cattle pasturalists, a predominantly male occupation, in areas where known vectors of kala-azar as well as lizard-feeding sandflies exist. Among the Anuak and Suri people, who live in areas of south-western Ethiopia infested with tsetse flies, the lizard trypanosome antigen gave a markedly higher skin test positivity than did the leishmanin antigen. This suggests that the lizard trypanosome may give artificially high results in other immunological tests of exposure to trypanosomiasis in areas where lizard trypanosomes and man-biting sandflies co-exist with mammalian trypanosomes transmitted by Glossina.     

The superiority of the miniature Anion-Exchange Centrifugation Technique for detecting low grade trypanosome parasitaemias

The efficiency was tested of the Haematocrit Centrifugation Technique (HCT) and the miniature Anion-Exchange Centrifugation Technique (mAECT) for demonstrating trypanosomes in the blood of four antelopes experimentally infected with Trypanosoma brucei gambiense in Liberia. During simultaneous daily application of both methods over a period of six months, parasitaemias were detected on 12 occasions by HCT and on 73 occasions by mAECT during 473 examination days, thus indicating the superiority of the mAECT for the detection of low grade trypanosome infections.     

Phylogenetic analysis reveals the presence of the Trypanosoma cruzi clade in African terrestrial mammals

Despite the impact of some trypanosome species on human and livestock health, the full diversity of trypanosomes in Africa is poorly understood. A recent study examined the prevalence of trypanosomes among a wide variety of wild vertebrates in Cameroon using species-specific PCR tests, but six trypanosome isolates remained unidentified. Here they have been re-examined using fluorescent fragment length barcoding (FFLB) and phylogenetic analysis of glycosomal glyceraldehyde phosphate dehydrogenase gGAPDH and 18S ribosomal RNA (rDNA) genes. Isolates from a monkey (Cercopithecus nictitans) and a palm civet (Nandinia binotata) belonged to the Trypanosoma cruzi clade, known previously only from New World and Australian terrestrial mammals, and bats from Africa, Europe and South America. Of the four other isolates, three from antelope were identified as Trypanosoma theileri, and one from a crocodile as T. grayi. This is the first report of trypanosomes of the T. cruzi clade in African terrestrial mammals and expands the clade's known global distribution in terrestrial mammals. Previously it has been hypothesized that African and New World trypanosomes diverged after continental separation, dating the divergence to around 100 million years ago. The new evidence instead suggests that intercontinental transfer occurred well after this, possibly via bats or rodents, allowing these trypanosomes to establish and evolve in African terrestrial mammals, and questioning the validity of calibrating trypanosome molecular trees using continental separation.     

An outbreak of Trypanosoma cruzi infection in Indian monkeys

Of a batch of ten Macacus rhesus monkeys received from India, the blood of four was later found to harbour trypanosomes. Two other monkeys of a previous batch, inoculated from an animal which subsequently died under suspicious circumstances before a trypanosome infection was suspected, also became infected.The infecting trypanosome was shown by morphological and cultural characters, as well as by its method of reproduction in tissues, to be indistinguishable from Trypanosoma cruzi.Transmission from one monkey to another by blood inoculations was possible in some cases, but one infected animal had never been inoculated.The source of the infection has not been definitely traced. It is known that the ship transporting the monkeys called at ports where T. cruzi infections occur in man, insects and animal carriers, and infection might thus have occurred during the journey. We cannot, however, completely exclude a laboratory infection.The possibility of T. cruzi infection should be borne in mind when working with rhesus monkeys.     

In Vitro Trypanocidal Activity of Antibodies to Bacterially Expressed Trypanosoma brucei Tubulin

Background

There are only four drugs for treating African trypanosomiasis, a devastating disease in sub-Saharan Africa. With slow discovery of better drugs, vaccination is viewed as the best method of control. We previously showed that antibodies to native Trypanosoma brucei brucei tubulin inhibit the growth of trypanosomes in culture. Here, we aimed to determine the effect of antibodies to bacterially expressed trypanosome tubulin on T. brucei brucei growth.

Methods

T. brucei brucei alpha and beta tubulin genes were individually expressed in Escherichia coli under the tryptophan promoter. Monoclonal tubulin antibodies reacted specifically with the expressed tubulins with no cross-reaction with the opposite tubulin. Rabbits were immunized with 450µg each of the concentrated recombinant tubulin, and production of antibodies assessed by ELISA and Western blotting. The effect of polyclonal antibodies on trypanosome growth was determined by culturing bloodstream T. brucei brucei in up to 25% of antisera.

Results

Low antisera dilutions (25%) from the immunized rabbits inhibited trypanosome growth. The most cytotoxic antisera were from one rabbit immunized with a mixture of both alpha and beta tubulins. However, the result was not reproduced in other rabbits and there was no apparent effect on growth at higher antisera dilutions.

Conclusion

Antibodies to bacterially expressed trypanosome tubulin are not effective at killing cultured bloodstream trypanosomes.     

Plasmodium falciparum in continuous culture: a new medium for the in vitro test for sulfadoxine sensitivity

The sulfadoxine sensitivity of two strains of Plasmodium falciparum from Thailand, FCM2 and FCM5, was assessed using two types of culture medium, Waymouth formula and RPMI 1640. Growth of the parasite was completely inhibited by 0.5 mmol of sulfadoxine per litre of Waymouth formula, whereas parasite growth in RPMI was not affected at this concentration. The apparent difference in drug sensitivity was shown to be caused by competition between 4-aminobenzoic acid and sulfadoxine. This hypothesis was further confirmed by the extent to which [¹⁴C]-sulfadoxine was incorporated into the infected erythrocytes.     

A silicone centrifugation technique for the detection of low parasitaemias of salivarian trypanosomes

A method using silicone fluid of specific gravity 1·075 was employed to detect low numbers of salivarian trypanosomes in rats infected with T. brucei, T. gambiense, T. congolense or mouse-adapted T. vivax. This method compared favourably with other microsensitive techniques such as the miniature anionexchange centrifugation and microhaematocrit buffycoat microscopy methods. The silicone centrifugation technique is based on the density differences between the host's erythrocytes and the parasites. Under the conditions used, the red cells are pelleted by centrifugation through a layer of silicone fluid whereas the trypanosomes remain in the plasma supernatant.     

Phylogenetic,morphological and behavioural analyses support host switching of Trypanosoma (Herpetosoma) lewisi from domestic rats to primates

We characterized four Brazilian trypanosomes isolated from domestic rats and three from captive non-human primates that were morphologically similar to T. lewisi, a considered non-pathogenic species restricted to rodents and transmitted by fleas, despite its potential pathogenicity for infants. These isolates were identified as T. lewisi by barcoding using V7V8 SSU rDNA sequences. In inferred phylogenetic trees, all isolates clustered tightly with reference T. lewisi and T. lewisi-like trypanosomes from Europe, Asia and Africa and despite their high sequence conservation formed a homogeneous clade separate from other species of the subgenus T. (Herpetosoma). With the aim of clearly resolving the relationships between the Brazilian isolates from domestic rats and primates, we compared sequences from more polymorphic ITS rDNA. Results corroborated that isolates from Brazilian rats and monkeys were indeed of the same species and quite close to T. lewisi isolates of humans and rats from different geographical regions. Morphology of the monkey isolates and their behaviour in culture and in experimentally infected rats were also compatible with T. lewisi. However, infection with T. lewisi is rare among monkeys. We have examined more than 200 free-ranging and 160 captive monkeys and found only three infected individuals among the monkeys held in captivity. The findings of this work suggest that proximity of monkeys and infected rats and their exposure to infected fleas may be responsible for the host switching of T. lewisi from their natural rodent species to primates. This and previous studies reporting T. lewisi in humans suggest that this trypanosome can cause sporadic and opportunistic flea-borne infection in primates.     

Tsetse flies: Genetics,evolution, and role as vectors

Tsetse flies (Diptera: Glossinidae) are an ancient taxon of one genus, Glossina, and limited species diversity. All are exclusively haematophagous and confined to sub-Saharan Africa. The Glossina are the principal vectors of African trypanosomes Trypanosoma sp. (Kinetoplastida: Trypanosomatidae) and as such, are of great medical and economic importance. Clearly tsetse flies and trypanosomes are coadapted and evolutionary interactions between them are manifest. Numerous clonally reproducing strains of Trypanosoma sp. exist and their genetic diversities and spatial distributions are inadequately known. Here I review the breeding structures of the principle trypanosome vectors, G. morsitans s.l., G. pallidipes, G. palpalis s.l. and G. fuscipes fuscipes. All show highly structured populations among which there is surprisingly little detectable gene flow. Rather less is known of the breeding structure of T. brucei sensu lato vis à vis their vector tsetse flies but many genetically differentiated strains exist in nature. Genetic recombination in Trypanosoma via meiosis has recently been demonstrated in the laboratory thereby furnishing a mechanism of strain differentiation in addition to that of simple mutation. Spatially and genetically representative sampling of both trypanosome species and strains and their Glossina vectors is a major barrier to a comprehensive understanding of their mutual relationships.