RESUMO
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.
Assuntos
Doenças dos Bovinos/parasitologia , Trypanosoma congolense , Tripanossomíase Africana/veterinária , Moscas Tsé-Tsé/parasitologia , Animais , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/epidemiologia , África do Sul/epidemiologia , Tripanossomíase Africana/sangue , Tripanossomíase Africana/epidemiologia , Tripanossomíase Africana/parasitologiaRESUMO
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.
Assuntos
Trypanosoma congolense , Tripanossomíase Africana/veterinária , Tripanossomíase Bovina/epidemiologia , Moscas Tsé-Tsé/parasitologia , Animais , Bovinos , DNA de Protozoário/análise , Genótipo , Camundongos , Parasitemia/epidemiologia , Parasitemia/parasitologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , África do Sul/epidemiologia , Trypanosoma congolense/classificação , Trypanosoma congolense/genética , Trypanosoma congolense/isolamento & purificação , Trypanosoma congolense/patogenicidade , Tripanossomíase Africana/epidemiologia , Tripanossomíase Africana/parasitologia , Tripanossomíase Bovina/parasitologiaRESUMO
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.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários , Camelus/parasitologia , Proteínas de Protozoários , Trypanosoma/imunologia , Tripanossomíase Africana/diagnóstico , Animais , Camelus/imunologia , Ensaio de Imunoadsorção Enzimática , Testes de Fixação do Látex , Valor Preditivo dos Testes , Proteínas Recombinantes , Tripanossomíase Africana/imunologiaRESUMO
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.
Assuntos
Antígenos de Protozoários/análise , DNA de Protozoário/sangue , Trypanosoma brucei brucei/isolamento & purificação , Trypanosoma vivax/isolamento & purificação , Tripanossomíase Bovina/diagnóstico , Moscas Tsé-Tsé/parasitologia , Animais , Bovinos , DNA de Protozoário/química , Ensaio de Imunoadsorção Enzimática/veterinária , Parasitemia/veterinária , Reação em Cadeia da Polimerase/veterinária , Sensibilidade e Especificidade , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/imunologia , Trypanosoma vivax/genética , Trypanosoma vivax/imunologia , Tripanossomíase Africana/diagnóstico , Tripanossomíase Africana/parasitologia , Tripanossomíase Africana/veterinária , Tripanossomíase Bovina/sangue , Tripanossomíase Bovina/parasitologiaRESUMO
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.
Assuntos
Antígenos de Protozoários/biossíntese , Proteínas de Protozoários/biossíntese , Trypanosoma/imunologia , Tripanossomíase/veterinária , Testes de Aglutinação/veterinária , Animais , Anticorpos Antiprotozoários/sangue , Variação Antigênica/imunologia , Antígenos de Protozoários/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Coelhos , Tripanossomíase/diagnóstico , Tripanossomíase/imunologiaRESUMO
Genetic analysis of Trypanosoma spp. depends on the detection of variation between strains. We have used the amplified fragment length polymorphism (AFLP) technique to develop a convenient and reliable method for genetic characterization of Trypanosome (sub)species. AFLP accesses multiple independent sites within the genome and would allow a better definition of the relatedness of different Trypanosome (sub)species. Nine isolates (3 from each T. brucei subspecies) were tested with 40 AFLP primer combinations to identify the most appropriate pairs of restriction endonucleases and selective primers. Primers based on the recognition sequences of EcoRI and BglII were chosen and used to analyse 31 T. brucei isolates. Similarity levels calculated with the Pearson correlation coefficient ranged from 15 to 98%, and clusters were determined using the unweighted pair-group method using arithmetic averages (UPGMA). At the intraspecific level, AFLP fingerprints were grouped by numerical analysis in 2 main clusters, allowing a clear separation of T. b. gambiense (cluster I) from T. b. brucei and T. b. rhodesiense isolates (cluster II). Interspecies evaluation of this customized approach produced heterogeneous AFLP patterns, with unique genetic markers, except for T. evansi and T. equiperdum, which showed identical patterns and clustered together.