Prevalence of mixed Trypanosoma congolense infections in livestock and tsetse in KwaZulu-Natal, South Africa.

Trypanosoma congolense causes the most economically important animal trypanosomosis in Africa. In South Africa, a rinderpest pandemic of the 1890s removed many host animals, resulting in the near-eradication of most tsetse species. Further suppression was achieved through spraying with dichlorodiphenyltrichloroethane (DDT); however, residual populations of Glossina austeni and G. brevipalpis remained in isolated pockets. A total of 506 of these tsetse flies were captured in the Hluhluwe-iMfolozi Park, the St Lucia Wetland Park and Boomerang commercial farm. The polymerase chain reaction (PCR) was used to determine the infection rate and frequency of mixed infections of these flies. Additionally, 473 blood samples were collected from cattle at communal diptanks and a commercial farm in the area and each one examined by the haematocrit centrifugation technique (HCT). Furthermore, buffy coats from these blood samples were spotted onto FTA Elute cards and the DNA extracted from each one tested using 3 separate PCRs. The HCT revealed the presence of trypanosomes in only 6.6% of the blood samples; by contrast, species-specific PCR detected trypanosome DNA in 50% of the samples. The species-specific PCR detected trypanosome DNA in 17% of the tsetse flies, compared with the nested PCR targeting rDNA which detected trypanosome DNA in only 14% of the samples. Over time, the transmission of Savannah-type T. congolense and Kilifi-type T. congolense as mixed infections could have an impact on disease manifestation in different hosts in the area.

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.

Recombinant RoTat 1.2 variable surface glycoprotein as antigen for diagnosis of Trypanosoma evansi in dromedary camels.

The transcript encoding a predominant Trypanosoma evansi variable surface glycoprotein RoTat 1.2 was cloned and expressed as a recombinant protein in Spodoptera frugiperda and Trichoplusia ni (insect) cells. Its potential as an antigen for specific detection of antibody in serum of dromedary camels affected by surra, was evaluated. In ELISA, the reactivity of the recombinant RoTat 1.2 VSG was similar to that of native RoTat 1.2 VSG. An indirect agglutination reagent was therefore prepared by coupling the recombinant RoTat 1.2 VSG onto latex particles. The performance of the latex agglutination test was evaluated on camel sera, and compared with the performance of CATT/T. evansi and LATEX/T. evansi tests, using the immune trypanolysis assay with T. evansi RoTat 1.2 as a reference test. The relative sensitivity and specificity of the latex coated with recombinant RoTat 1.2 VSG, using a 1:4 serum dilution, were respectively, 89.3 and 99.1%. No differences were observed between the performance of latex coated with recombinant RoTat 1.2 VSG and LATEX/T. evansi or CATT/T. evansi. Here, we describe the successful use of the recombinant RoTat 1.2 VSG for detection of specific antibodies induced by T. evansi infections.

Analysis of erythropoietin and erythropoietin receptor genes expression in cattle during acute infection with Trypanosoma congolense.

Acute Trypanosoma congolense infection induced moderate, transient anemia in N'Dama cattle (trypanotolerant) and severe anemia in Boran cattle (trypanosusceptible). Erythropoietin receptor (EpoR) was cloned and sequenced from the two breeds of cattle. A single position mutation of Tyr in the Boran to His in the N'Dama predicted amino acid sequence was revealed. The mRNA transcription of erythropoietin (Epo) in kidneys and EpoR in the bone marrow of infected cattle was determined by competitive reverse transcription and the polymerase chain reaction (RT-PCR). Though Epo mRNA transcription increased in the kidneys during infection, the increase was not significantly different (p>0.05) between the two breeds of infected cattle. The level of EpoR transcripts in the bone marrow of infected N'Damas was significantly higher (p<0.05) than that detected in the marrows from infected Boran cattle. While infection seem to increase levels of transcription of IL-1alpha and beta, and TNFalpha in kidneys from both Boran and N'Dama cattle, no significant difference was detected in the level of mRNAs of these cytokines in the kidney from the two breed of cattle. The amount of IFNgamma mRNA transcripts were not changed with infection in N'Dama cattle, while on the contrary a significant higher levels of IFNgamma was found in kidneys from infected Boran cattle as compared to the other groups. A significant (p<0.05) increase in the levels of IL-1alpha and beta, and IFNgamma mRNA transcripts were detected in the marrows of infected Borans as compared to the infected N'Dama cattle. In this study the increase in the level of TNFalpha mRNA in the marrows of the two infected breeds was not different. This implies there is no negative effect of TNFalpha on hematopoiesis during acute infection. These findings suggest that the levels of Epo and EpoR in the infected Boran cattle were inadequate for their degree of anemia, which might be due in part to high expression of IFNgamma during acute infection with T. congolense.

Generation of expressed sequence tags as physical landmarks in the genome of Trypanosoma brucei.

Previous molecular genetic studies on the African trypanosome have focused on only a few genes and gene products, the majority of which are concerned with surface antigenic variation; consequently, an insignificant number of the genes of this organism have been characterized to date. In order to: (1) identify new genes and analyze their expression profile, (2) generate expressed sequence tags (ESTs) for derivation of a physical map of the trypanosome genome, and (3) make available the partial sequence information and the corresponding clones for general biomedical research on the parasite, we have performed single-pass sequencing of random, directionally cloned cDNAs from a bloodstream form Trypanosoma brucei rhodesiense library. Analysis of 2128 such ESTs sequenced so far in this study showed significant similarities [BLASTX P(n)-value < 10(-4), and a match > 10 amino acid residues] with proteins whose genes have been described in diverse organisms including man, rodents, kinetoplastids, yeasts and plants. A number of the ESTs encode homologues of proteins involved in various functions including signal reception and transduction, cell division, gene regulation, DNA repair and replication, general metabolism, and structural integrity. Although some of these genes may have been expected to be present in the African trypanosomes, the majority of them had not previously been described in these organisms. A large proportion, 768 individual ESTs (36%, representing 385 different transcripts), had a significant homology with genes described in organisms other than the African trypanosomes; however, 15% of the ESTs were from genes already described in trypanosomes. Among the ESTs analysed were 462 distinct known genes, only 77 of which have been described in T. brucei. Approximately 52% of the ESTs did not show any significant homology with the sequences in any of the public domain databases.

Minichromosomal variable surface glycoprotein genes and molecular karyotypes of Trypanosoma (Nannomonas) congolense.

Employing orthogonal-field-alternation gel electrophoresis (OFAGE), we have separated chromosome-sized DNA molecules from Trypanosoma (Nannomonas) congolense clones, the clones being derived from several distinct antigenic repertoires. Trypanosome clones that belong to a specific antigenic repertoire appear to have a chromosome pattern characteristic of that particular repertoire. Hybridization of the separated chromosomes with cloned DNA fragments encoding variable surface glycoproteins revealed the presence of two different T.(N.) congolense variable surface glycoprotein genes on mini-chromosomes (mc) and the modes by which these genes may be activated: one by duplicative and the other by non-duplicative activation.

Metacyclic form-specific variable surface glycoprotein-encoding genes of Trypanosoma (Nannomonas) congolense.

A complementary DNA expression library in phage lambda gt11 was synthesized using mRNA from in vitro-produced metacyclic forms of a clone of Trypanosoma (Nannomonas) congolense. The unamplified library was screened with antiserum from a goat immune to infection with metacyclic (m)-forms of T. congolense ILRAD Nannomonas antigen repertoire 2(ILNaR2). Of the 100 antiserum-reactive phage clones identified, 22 were analyzed further: 21 of the clones contained overlapping portions of a single transcript, while one other contained a different transcript. Northern blot analyses indicated that the sequences contained in the clones were transcribed only by m-forms of ILNaR2. Immunological and sequence analyses indicated that the two different cloned sequences encode m-form-specific variable surface glycoproteins.

Cloning of a cDNA encoding bovine erythropoietin and analysis of its transcription in selected tissues.

A bovine cDNA encoding erythropoietin (Epo) was isolated by polymerase chain reaction (PCR) amplification and screening of a bovine kidney cDNA library. The sequenced cDNA has a length of 1312 bp and an open reading frame that encodes a predicted 192-amino-acid (aa) protein, including a putative signal sequence of 25 aa. A mature protein of 167 aa (18.4 kDa) results upon cleavage of the putative signal peptide. The deduced bovine mature Epo peptide exhibits 96, 88, 83, 82 and 79% sequence identity to that of sheep, swine, human, monkey and rat, respectively. The expression of the bovine Epo gene in tissues from a severely anemic calf, bovine fetus and a healthy steer was analysed by a competitive RT-PCR method. In kidneys of the severely anemic calf, Epo mRNA levels increased 60-fold relative to that from the kidneys of the healthy steer. Epo mRNA levels were threefold higher in the liver of the bovine fetus than that in its kidneys. Low levels of Epo transcripts were detected in RNA from spleen of the severely anemic calf and the bovine fetus. No Epo transcripts were detectable in spleen from the healthy steer.

Recombinant DNA probes reveal simultaneous infection of tsetse flies with different trypanosome species.

The utility of recombinant DNA probes in the detection of natural trypanosome infection of tsetse flies has been assessed in Lambwe Valley, near the shores of Lake Victoria, Kenya. The tsetse flies were surveyed during two different seasons in 1988. Three different probes used each contained highly repetitive DNA sequences specific for a species or subspecies of trypanosomes of the Nanomonas subgenus. A fourth probe contained repetitive sequences common to trypanosome species of the Trypanozoon subgenus. Mixed mature or immature infections were detected in a variety of combinations in different individual tsetse flies. Such infections were detected in both the guts and mouthparts of some tsetse flies. Simultaneous natural infection of tsetse with the savannah type Trypanosoma congolense and Kilifi type T. congolense, T. congolense and Trypanosoma brucei or T. congolense and Trypanosoma simiae were demonstrated. The probes have thus been used to demonstrate the presence of Lambwe Valley, south-western Kenya, of a type of T. congolense first observed among trypanosome isolates obtained from sentinel cattle exposed to natural infection on a ranch at Kilifi on the Kenya coast. This type of T. congolense appears not to be confined to the coastal region nor to any particular species of tsetse flies and may contribute significantly to livestock morbidity in other areas of eastern Africa. In the Kilifi area, T. congolense was found primarily in Glossina austeni; in Lambwe valley, it was found in Glossina pallidipes.

Cloning and analysis of Trypanosoma (Nannomonas) congolense ILNat 2.1 VSG gene.

Genes encoding various Trypanosoma (Trypanozoon) brucei variable surface glycoproteins (VSGs) show considerable conservation among different members of this species, known as isotypes. The occurrence of isotypes in other salivarian trypanosomes has not been well documented. We have cloned sequences encoding Trypanosoma (Nannomonas) congolense ILNat 2.1 VSG, and used it in DNA blot hybridization analyses of this and other T. congolense clones originating from geographically separate regions of East Africa. The data indicate that the expression of ILNat 2.1 VSG gene proceeds by duplicative transposition resulting in the presence of an extra expression-linked copy in the expressing clones examined. Furthermore the ILNat 2.1 VSG gene sequence is absent or has greatly diverged, in all other T. (N.) congolense clones that belong to different serodemes. This suggests that some T. (N.) congolense VSGs may be limited to their respective antigen repertoires. The data are discussed in the light of their implications for antigenic variation in T. (N.) congolense, and parasite epidemiology.

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).

Analyses of variable antigen gene rearrangements in Trypanosoma brucei.

For the purpose of investigating the genetic basis of antigenic variation in Trypanosoma brucei, we have analyzed the structure of the genome surrounding the gene coding for one T. brucei variable antigen (ILTat 1.2) in several T. brucei clones expressing this and other variable antigens. In each case there are two copies of the gene. We found no evidence of an extra copy associated with the expression of this gene. Differences were found between the two copies in a single clone, and between the copies in different clones. The differences could be explained by insertion and deletion of various lengths of DNA in a region beyond the C-terminal end of the gene. Differences in genomic structure were found even between clones expressing the same antigen, whether ILTat 1.2 or another. Thus, no feature of the rearrangements observed can be correlated with the expression of a particular antigen.

Long-term occurrence of Trypanosoma congolense resistant to diminazene, isometamidium and homidium in cattle at Ghibe, Ethiopia.

Ten trypanosome isolates were collected at random from cattle at Ghibe, Ethiopia, in February 1993 and all shown to be savannah-type Trypanosoma congolense. When inoculated into naïve Boran (Bos indicus) calves, all 10 isolates were resistant to diminazene aceturate (Berenil), isometamidium chloride (Samorin) and homidium chloride (Novidium) at doses of 7.0 mg/kg body weight (b.w.), 0.5 mg/kg b.w. and 1.0 mg/kg b.w., respectively. In order to determine whether this multiple-drug resistance was expressed by individual trypanosomes, clones were derived from two of the isolates and characterised in mice for their sensitivity to the three compounds; by comparison to drug-sensitive populations, the two clones expressed high levels of resistance to all 3 trypanocides. In experiments to characterise the uptake kinetics of [14C]-Samorin, the maximal rates of uptake (Vmax) for 4 Ghibe isolates ranged from 9.2 to 15.0 ng/10(8) trypanosomes/min. In contrast, Vmax for the isometamidium-sensitive clone T. congolense IL 1180 was 86.7 +/- 8.6 ng/10(8) trypanosomes/min. Lastly, molecular karyotypes were determined for eight isolates: seven different chromosome profiles were observed. These data indicate that in February 1993 there was a high prevalence of drug-resistant trypanosome populations with different chromosome profiles in cattle at Ghibe. Since a similar situation existed at the same site in July 1989, this suggests that the drug-resistance phenotype of trypanosomes at Ghibe had not altered over a 4 year period.

Epidemiology of bovine trypanosomiasis in the Ghibe valley, southwest Ethiopia. 3. Occurrence of populations of Trypanosoma congolense resistant to diminazene, isometamidium and homidium.

In July 1989, blood samples were collected from parasitaemic cattle in the Ghibe valley, Ethiopia, frozen in liquid nitrogen and transported to Nairobi, Kenya. Twelve of the stabilates were inoculated into individual Boran (Bos indicus) calves and characterised for their sensitivity, in turn, to diminazene aceturate (Berenil), isometamidium chloride (Samorin) and homidium chloride (Novidium). All 12 stabilates produced infections which were shown to be Trypanosoma congolense and resistant to treatment with diminazene aceturate at a dose of 7.0 mg kg-1 body weight (b.w.). Eleven of the infections were also resistant to isometamidium chloride at a dose of 0.5 mg kg-1 b.w. and homidium chloride at a dose of 1.0 mg kg-1 b.w. The drug-sensitivity phenotypes of three of the same isolates were also determined in goats which were each treated with only one of the three trypanocides: all expressed the same phenotypes as the populations expressed in the aforementioned Boran calves. Five clones were derived from one of the isolates which expressed a high level of resistance to all three trypanocides; each clone expressed high levels of resistance to all three trypanocides when characterised in mice. Thus, the multi-resistance phenotype of the parental isolate was associated with expression of mutli-resistance by individual trypanosomes. Finally, molecular karyotypes and electrophoretic variants of six enzymes were determined for seven and eight of the isolates, respectively. Six different karyotypes were observed and all eight of the latter isolates belonged to different zymodemes, indicating that the multi-resistance phenotype at Ghibe was associated with many genetically distinct populations.

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.

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.

The expression of RoTat 1.2 variable surface glycoprotein (VSG) in Trypanosoma evansi and T. equiperdum.

In order to define whether the variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi.

The application of molecular biological tools to epidemiology of African trypanosomosis.

Difficulties have often been encountered in the field surveys due to a lack of definitive morphological characters, particularly where mixed infections are expected. To address this problem, some molecular biological techniques such as DNA probe hybridization, restriction fragment length polymorphism (RFLP) analysis, the polymerase chain reaction (PCR), analyses of ribosomal DNA, and pulsed-field gel electrophoresis (PFGE), have been applied to the analysis of field samples collected during epidemiological surveys of African trypanosomosis. Concurrent natural infection of different individual tsetse flies and mammalian hosts with different species of the trypanosomes have been demonstrated, through the use of a combination of specific DNA probe hybridization and the PCR. Molecular karyotypes of Trypanosoma brucei species were analyzed by PFGE in 45 - 2,000 kb range. There are distinctive differences in intermediate and mini-chromosomes among the strains. We have compared the nucleotide sequences of ribosomal DNAs of the parasites by PCR techniques. From this data new phylogenetic tree can be inferred. It is apparent that these technologies can provide powerful tools for identification and diagnosis of trypanosomes in their hosts and vectors, and for their more accurate phylogenetic classification.

A repetitive deoxyribonucleic acid sequence distinguishes Trypanosoma simiae from T. congolense.

The dominant repetitive deoxyribonucleic acid (DNA) sequence in the genome of a clone of Trypanosoma (Nannomonas) simiae has been identified and cloned as a recombinant plasmid. The recombinant plasmid was used in hybridization analyses of DNA samples obtained from various trypanosome species and subspecies. The results indicated that the T. simiae repetitive DNA sequence hybridized with DNA derived only from T. simiae; it did not hybridize with DNA derived from clones or stocks of T. congolense, or from any other trypanosome species examined. A preliminary characterization of the cloned DNA sequence and its use in the identification of T. simiae of similar genotypes are presented.

Physical and transcriptional organization of the ribosomal RNA genes of the savannah-type Trypanosoma congolense.

Ribosomal RNA genes have been cloned from the major species of African trypanosomes. Complete nucleotide sequence composition of the small subunit (SSU) and portions of the large subunit (LSU) ribosomal RNA genes was determined for each of these trypanosome species. In contrast to the situation in Trypanosoma brucei, in savannah-type T. congolense the LSU ribosomal RNA is cleaved twice, to generate two additional prominent fragments. This leads to the different profiles observed when the rRNA molecules from these two trypanosome species are resolved in agarose gels. From the nucleotide sequences of the 18S RNA, a phylogenetic tree was derived depicting the relationships among the T. congolense complex of trypanosomes and the other species of trypanosomes.