Apparent lack of a domestic animal reservoir in Gambiense sleeping sickness in northwest Uganda

The role played by domestic animals in the transmission of gambiense Human African Trypanosomosis remains uncertain. Northwest Uganda is endemic for Trypanosoma brucei gambiense. Of the 3267 blood samples from domestic animals in four counties examined by hematocrit centrifugation technique (HCT), 210 (6.4%) were positive for trypanosomes. The prevalence of animal trypanosomosis was estimated at 13.8% in Terego County, 4.2% in East Moyo County, 3.1% in Koboko County, and zero in West Moyo County. The trypanosome infection rates varied from 0.2% in goats, 3.5% in dogs, 5.0% in sheep, 7.5% in cattle, to 15.5% in pigs. DNA was extracted from the blood samples by Chelex method, Sigma and Qiagen DNA extraction Kits. A total of 417(12.8%) DNA samples tested positive by polymerase chain reaction (PCR) using T. brucei species specific primers (TBR) indicating that the DNA was of Trypanozoon trypanosomes while 2850 (87.2%) samples were TBR-PCR negative. The T. brucei infection rates based on TBR-PCR were highest in pigs with 21.7%, followed by cattle (14.5%), dogs (12.4%), sheep (10.8%), and lowest in goats with 3.2%, which indicated that pigs were most bitten by infected tsetse than other domestic animals. TBR-PCR detected 6.3% more infected domestic animals that had been missed, and confirmed the 6.4% cases detected by HCT in the field. Statistical analysis done using one-way ANOVA Kruskal-Wallis test (Prism version 5.0) showed no significant difference in trypanosome infections among domestic animals using both HCT and TBR-PCR techniques in the different counties (Confidence Interval of 95%, p-values >0.05). All the 417 trypanosome DNA samples were negative by PCR using two sets of primers specific for the T. b. gambiense specific glycoprotein gene and serum resistance associated gene of T. b. rhodesiense, indicating that they were probably not from the two human infective trypanosomes. Polymerase chain reaction using primers based on ribosomal internal transcribed spacer-1 region (ITS-PCR) resolved the 417 DNA of trypanosome samples into 323 (77.5%) as single trypanosome infections due to T. brucei and 39 (9.4%) mixed infections but missed detecting 55 (13.1%) samples, possibly because of the low sensitivity of ITS-PCR as compared to TBR-PCR. The 31 mixed infections were due to T. brucei (T.b) and T. vivax (T.v); while 8 mixed infections were of T. congolense (T.c) and T. brucei but no mixed trypanosome infections with T. congolense, T. brucei, and T. vivax were detected. Statistical analysis done using one way ANOVA Kruskal-Wallis test (Prism version 5.0) to compare single and mixed trypanosome infections showed no significant difference in trypanosome infections due to single (T.v, T.b, T.c) and mixed (T.v+T.b; T.v+T.c; T.b+T.c; T.v+T.b+T.c) trypanosome species among domestic animals in the different counties using ITS-PCR technique (Confidence Interval of 95%, p-values >0.05). It was concluded that domestic animals in northwest Uganda were probably not reservoirs of T. b. gambiense and there was no infection, as yet, with T. b. rhodesiense parasites.     

The effect of flurbiprofen, a potent non-steroidal anti-inflammatory agent, upon Trypanosoma vivax infection in goats

Platelet aggregation leading to a decreased number of thrombocytes and reduced blood serotonin levels can be correlated with parasitaemia as has been observed in goats and cattle infected with T. vivax Y58. Flurbiprofen is a potent anti-inflammatory agent with antipyretic activity. In vitro , this agent inhibits platelet aggregation and blocks serotonin release. The results of the present study demonstrated that flurbiprofen inhibited the febrile reactions during the acute phase of T. vivax infection, but the drug did not prevent or reverse the associated drop in blood serotonin level during this period. Moreover, it was apparent that flurbiprofen had a deleterious effect on goats infected with T. vivax Y58. The infection in the untreated animals (sixteen out of seventeen goats) followed a rather mild and prolonged course with peaks of parasitaemia during the febrile episodes, whereas in flurbiprofen-treated goats (five animals), inoculated with the same number of trypanosomes, the parasitaemia was progressive and terminated in early death with disseminated intravascular coagulation at post mortem examination. These observations would seem to confirm the work of previous investigators, suggesting that anti-inflammatory agents have an aggravatory effect on the course of infection in animals inoculated with various strains of trypanosomes. Important differences exist, however, in the relationship between prostaglandin synthesis in the platelets of the goat and in those of other species.     

Effect of chemotherapy on elevated ejaculation time and deteriorated semen characteristics consequent to bovine trypanosomiasis

The effect of the trypanocidal drug Novidium on elevated ejaculation time and deteriorated semen characteristics was studied in Zebu cattle infected with T. vivax and T. congolense. Two groups, comprising six bulls per group, were infected with Trypanosoma vivax or Trypanosoma congolense while three bulls served as controls. Chemotherapy was carried out 12 weeks post-infection on three bulls from each group, leaving three bulls untreated while three bulls served as uninfected controls. Blood samples from treated bulls were all negative for trypanosomes 3 days post-chemotherapy. The animals also had normal body temperature. As the study progressed, clinical signs associated with trypanosomiasis, such as anaemia and cachexia, disappeared gradually in treated bulls. There was some improvement in semen characteristics of some of the bulls at 10 weeks post-chemotherapy with Novidium. However, all bulls infected with T. vivax or T. congolense irrespective of Novidium chemotherapy still had poor semen characteristics manifested by all or some of the following: decreased volume of semen, oligospermia, azoospermia and elevated incidence of spermatozoa morphological abnormalities. They were thus unsuitable for breeding.     

Enzootiology of Trypanosoma evansi in Pantanal, Brazil

In order to better understand the enzootiology of trypanosomiasis caused by Trypanosoma evansi in the Brazilian Pantanal we examined domestic and wild mammals by microhematocrit centrifuge technique (MHCT), immunofluorescence antibody test (IFAT) and polymerase chain reaction (PCR). T. evansi infection was detected in all species sampled with exception of the sheep and the feral pig. High parasitemias were observed in capybaras (5/24), coatis (18/115), horses (31/321) and dogs (3/112). Among these species, only the capybaras did not develop anemia. Low parasitemias, only detected by PCR, were found in buffaloes (18/43), bovines (29/331), marsupials (1/4), small rodents (14/67), bats (7/18), and one armadillo (1/8). The highest prevalence of T. evansi infection was recorded in horses (73%), although no neurological signs in infected horses were observed. Diagnosis through standard parasitological tests and IFAT should be used with caution since they may overlook comprovedly infected horses. The relationship between ranch management and T. evansi infection in horse was investigated. The importance of other transmission mechanisms apart from the tabanids and reservoir hosts are discussed.     

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.     

Genetic analysis of Trichuris suis and Trichuris trichiura recovered from humans and pigs in a sympatric setting in Uganda

The whipworms Trichuris trichiura and Trichuris suis in humans and pigs, respectively, are believed to be two different species yet closely related. Morphologically, adult worms, eggs and larvae of the two species are indistinguishable. The aim of this study was to examine the genetic variation of Trichuris sp. mainly recovered from natural infected pigs and humans. Worm material isolated from humans and pigs living in the same geographical region in Uganda were analyzed by PCR, cloning and sequencing. Measurements of morphometric characters were also performed. The analysis of the ITS-2 (internal transcribed spacer) region showed a high genetic variation in the human-derived worms with two sequence types, designated type 1 and type 2, differing with up to 45%, the type 2 being identical to the sequence found in pig-derived worms. A single human-derived worm showed exclusively the type 2-genotype (T. suis-type) and three cases of 'heterozygote' worms in humans were identified. However, the analysis showed that sympatric Trichuris primarily assorted with host origin. Sequence analysis of a part of the genetically conserved β-tubulin gene confirmed two separate populations/species but also showed that the 'heterozygote' worms had a T. suis-like β-tubulin gene. A PCR-RFLP on the ITS-2 region was developed, that could distinguish between worms of the pig, human and 'heterozygote' type. The data suggest that Trichuris in pigs and humans belong to two different populations (i.e. are two different species). However, the data presented also suggest that cross-infections of humans with T. suis takes place. Further studies on sympatric Trichuris populations are highly warranted in order to explore transmission dynamics and unravel the zoonotic potential of T. suis.     

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.     

Evaluation of Diagnostic Tests for <Emphasis Type="Italic">Trypanosoma evansi</Emphasis> in Experimentally Infected Pigs and Subsequent Use in Field Surveys in North Vietnam and Thailand

This study is concerned with the evaluation of established diagnostic tests for diagnosis of Trypanosoma evansi in pigs. The immune trypanolysis test (TL), card agglutination test (CATT), latex agglutination test (LATEX), enzyme-linked immunosorbent assay (ELISA), microhaematocrit centrifugation technique (MHCT) and mouse inoculation (MI) tests were initially evaluated in experimentally infected fattening pigs. All infected pigs were confirmed parasitologically positive with both MHCT and MI. Results of the serological assays indicated that the TL could be a reference test for the presence of RoTat 1.2 antibodies in pigs. The results of the CATT and LATEX were inconsistent with the TL while the ELISA results correlated with the TL results. The four serological assays were subsequently used in two field surveys in Vietnam and Thailand. Results of the two agglutination assays (CATT and LATEX) were not consistent and did not correlate with TL results. The ELISA at percentage positivity of 22 appeared to have good ability to discriminate between seropositive and seronegative animals. Of the 437 samples collected at smallholder pig premises in northern Vietnam, no positive pigs were detected with the TL test. In Thailand, 77 samples were collected from five farrowing farms with a history of surra. Two parasitologically positive sows were found and on each farm seropositive sows were detected.     

The application of PCR-ELISA to the detection of Trypanosoma brucei and T. vivax infections in livestock

Teneral tsetse flies infected with either Trypanosoma brucei or T. vivax were fed on healthy cattle. Blood samples collected daily from the cattle were examined by microscopy for the presence of trypanosomes, in thick smear, thin smear and in the buffy coat (BC). All the cattle fed upon by infected tsetse developed a fluctuating parasitaemia. DNA was extracted from the blood of these cattle and subjected to polymerase chain reaction (PCR) using oligonucleotide primers specific for T. brucei or T. vivax. The PCR products unique to either T. brucei or T. vivax were identified following amplification of DNA from the blood samples of infected cattle, whereas none was detectable in the DNA from the blood of the cattle exposed to non-infected teneral tsetse. In a concurrent set of experiments, one of the oligonucleotide primers in each pair was biotinylated for use in PCR-ELISA to examine all the blood samples with this assay. Both the PCR and the PCR-ELISA revealed trypanosome DNA in 85% of blood samples serially collected from the cattle experimentally infected with T. brucei. In contrast, the parasitological assays showed trypanosomes in only 21% of the samples. In the blood samples from cattle experimentally infected with T. vivax, PCR and PCR-ELISA revealed trypanosome DNA in 93 and 94%, respectively. Microscopy revealed parasites in only 63% of the BCs prepared from these cattle. Neither PCR nor PCR-ELISA detected any trypanosome DNA in blood samples collected from the animals in the trypanosome-free areas. However, both assays revealed the presence of trypanosome DNA in a number of blood samples from cattle in trypanosomosis-endemic areas.     

Isolation of two antigens from Trypanosoma evansi that are partially responsible for its cross-reactivity with Trypanosoma vivax

In Venezuela, two non-tsetse transmitted trypanosomes, Trypanosoma evansi and Trypanosoma vivax, are the major etiological agents of animal trypanosomosis. Rodents can be experimentally infected with T. evansi in order to obtain enough parasites to prepare antigens for serological tests. On the contrary, the production of T. vivax antigens is a limiting factor in most laboratories. Since T. evansi and T. vivax have exhibited a very high immunological cross-reactivity, we have focused on the identification of antigens from T. evansi responsible for this phenomenon. The predominant 64 kDa glycosylated cross-reacting antigen was recently purified from the TEVA1 T. evansi Venezuelan isolate [Parasitology 124 (2002) 287]. Here, we purified two additional cross-reacting antigens with molecular masses of approximately 51 and 68 kDa from the cytosolic fraction of the same T. evansi isolate, by sequential chromatography on DEAE-sepharose and sephacryl S-300. Sera obtained from animals infected with T. evansi or T. vivax recognized both purified proteins, suggesting their potential use as diagnostic reagents.     

Prevalence and incidence of trypanosomosis in horses and donkeys in the Gambia

In a study of the prevalence and incidence of trypanosomosis in horses and donkeys in two regions of the Gambia, surveys were carried out at Niamina east and Bansang south with a high and low to moderate tsetse challenge, respectively. Eleven horses and 67 donkeys were sampled monthly from August 1997 to September 1998. Blood samples were examined for trypanosomes using the buffy-coat (BC) method and polymerase chain reaction (PCR). Three primer sets were used, specific for either Trypanosoma vivax (TVW), Trypanosoma congolense (GOL) or Trypanosoma brucei (ORPHON5J).The BC results showed that the prevalence (August 1997) and the average monthly incidence (September 1997-1998) of trypanosome infections in horses (45.5 and 16%, respectively) were significantly higher than in donkeys (6.2 and 9%, respectively). Using PCR, the number of detected cases was seven times higher than using the BC. T. congolense was the most frequently observed species, followed by T. vivax and T. brucei. This study confirms earlier observations by other authors that donkeys, which are exposed to a similar tsetse challenge as horses, are significantly less infected with trypanosomes than the latter.     

Biting flies and Trypanosoma vivax infection in three highland districts bordering lake Tana, Ethiopia

An epidemiological study was conducted to determine the prevalence of trypanosomosis in cattle, small ruminants and Equidae, and to identify biting flies; potential mechanical vectors of trypanosomes in the three districts of Bahir Dar Zuria, Dembia and Fogera, bordering lake Tana, Ethiopia. About 1509 cattle, 798 small ruminants and 749 Equidae were bled for the prevalence study using the buffy-coat method and the measurement of the hematocrit value. Sixty-six NGU and 20 monoconical traps were deployed for the fly survey. The results indicated the presence of trypanosomes in 6.1% (92/1509) of the cattle with a maximum during the late rainy season (9.6%) than the early dry season (3.6%) at Fogera district. Prevalence at the district level varied from 4% to 9.6%. Only one sheep (1/122) and one goat (1/676) were found positive for T. vivax-like trypanosomes and none of the Equidae was positive. All the trypanosomes encountered in cattle belong to the single species of T. vivax. The PCV was negatively associated with detection of T. vivax (21.6% in infected versus 25.4% in non-infected cattle). A total of 55,398 biting flies were caught of which 49,353 (89.08%) belong to Stomoxys, 4715 (8.51%) to horse flies and 1330 (2.4%) to Chrysops species. There was no tsetse fly. Species identification has indicated the presence of Atylotus agrestis, Chrysops streptobalia, Stomoxys calcitrans, S. nigra, S. pulla, S. pallida, S. sitiens, S. taeniata, S. uruma, Haematopota lasiops and Hippobosca variegata. The overall apparent density was 214.7flies/trap/day. Seasonal comparison showed higher fly catches in the late rainy season than the early dry season. This study indicated that T. vivax infections culminate in cattle at the same time as mechanical vectors such as Stomoxys sp. and Atylotus agrestis. Therefore, attention towards T. vivax infection in cattle is essential to control the impact of the disease on productivity. A further study on biting flies is recommended.     

The relationship between dietary energy levels and the severity of Trypanosoma brucei infection in growing pigs

Growing pigs were placed on high, medium and low planes of dietary energy and were infected with a virulent strain of Trypanosoma brucei. During an 8-week period post-infection (p.i.), the respective liveweight gains by infected pigs on high, medium and low energy levels were 52.1, 21.2 and 38.5%, respectively, of the corresponding gains by non-infected control pigs. There was a fall in red blood cell values p.i. which worsened with decreasing energy levels. Leucocytosis was observed in all infected pig groups and was mainly due to lymphocytosis. By 6 weeks p.i., the lymphocyte count had returned to near normal values in pigs on high and medium energy levels, but was persistently high in those on a low energy level. Neutropaenia was observed in all infected pig groups and persisted until 8 weeks p.i. The results indicated that nutrition modulates the host response to infection with trypanosomes.     

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.     

Effect of novidium (homidium chloride) chemotherapy on genital lesions induced by Trypanosoma vivax and Trypanosoma congolense infections in Zebu bulls

Zebu bulls chronically infected with Trypanosoma vivax and T. congolense were treated at the 12th week post-infection with novidium and slaughtered at different times after treatment to determine histological evidence of healing of the genital lesions. Though trypanosomes disappeared from the blood soon after chemotherapy, there was incomplete resolution of genital lesions even 10-18 weeks later. Where there is severe degeneration of the testes and epididymes chemotherapy may be ineffective in leading to regeneration and therefore infertility problems may persist in bulls with chronic trypanosomiasis which are later subjected to chemotherapy.     

Absence of host proteins from the surface of Trypanosoma vivax of cattle

Trypanosoma vivax (EATRO 1721) organisms were isolated by DEAE-cellulose chromatography from blood of an experimentally infected calf. Attempts to agglutinate the purified trypanosomes with a rabbit antiserum against whole bovine serum or antisera monospecific for bovine IgG1, IgG2, IgM, complement component C3 or albumin were unsuccessful. The trypanosomes, however, were agglutinated by immune sera of four different calves chronically infected with T. vivax (EATRO 1721). It was concluded that T. vivax organisms purified by DEAE-cellulose chromatography from blood of cattle do not have bovine serum proteins on their surface.     

Prevalence of gastrointestinal nematodes in growing pigs in Kabale District in Uganda

During the last 30 years, pig production in Uganda and neighbouring counties has increased markedly. Pigs are mainly kept as a source of income for small-scale farmers; however, the pig production is subject to several constraints, one of them being worm infections. A study was carried out in rural communities in Kabale District in the South Western part of Uganda in September and October 2007 in order to estimate the prevalence of gastrointestinal nematode parasites in pigs based on coprological examination. Fifty-six households were randomly selected and visited. Housing system and deworming history were recorded. Faeces was sampled from rectum of one to five pigs (age, 3–12 months) per household. A total of 106 pigs were examined coprologically of which 91% excreted nematode eggs. The following prevalences of nematode eggs were recorded: strongyles (89%), Ascaris suum (40%), Trichuris suis (17%) and spiruroid eggs (48%). On household level, rearing pigs on slatted floors in pens significantly reduced the faecal egg excretion of strongyle eggs with almost 80% (p = 0.010) and a significant interaction between floor type and anthelmintic treatment was found for spiruroids (p = 0.037). Fifteen T. suis egg positive pigs were selected for post-mortem examination of the gastrointestinal tract. The post-mortem examinations revealed that 93% pigs were infected with Oesophagostomum spp. (worm burden, min–max 10–2,180), 73% with A. suum (1–36), 67% with T. suis (6–58), and 20% with Hyostrongylus rubidus (worms not quantified). In general, nematode infections were widespread and polyparasitism common in pigs in Uganda. However, worm burdens were moderate which may be related to recent deworming or to the practice of rearing pigs on slatted floors in wooden elevated pens.     

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.     

Action of isometamidium chloride on the insect vector form of Trypanosoma vivax

The effect of isometamidium chloride upon developing and mature Trypanosoma vivax occurring in Glossina palpalis palpalis flies was evaluated. Newly emerged G p palpalis flies were infected with T vivax by allowing them to feed on parasitaemic animals. Two experiments were conducted and in each the flies were divided into two groups. One group of infected flies was fed in vitro through a membrane on defibrinated cow blood containing isometamidium chloride at 0.1 mg ml-1, after which they were dissected and examined for trypanosomes, and the other group was fed in the same way on unmedicated blood. The results showed that out of a total number of 129 flies which fed on medicated blood, none was infected, while 55 out of 127 flies which fed on unmedicated blood were infected. The results indicate that isometamidium chloride eliminated the insect vector form of T vivax. These findings are of potential significance in the control of trypanosomiasis in the field, particularly in the operation of the sterile insect technique.     

Pathology of Experimental Trypanosomiasis in the Albino Rat, Rabbit, Goat and Sheep — A Preliminary Report

In rats, rabbits, sheep, and goats experimentally infected with several strains of Trypanosoma brucei, the trypanosomes were observed to localise extravascularly in connective tissues. Focal inflammatory reactions were associated with the localisation of the parasites. Trypanosoma congolense in the same species of animals and T. vivax in sheep and goats, were not observed to localise outside blood vessels. On the basis of these observations it appears that the pathogenesis of the disease caused by T. brucei differs from that caused by T. congolense and T. vivax.