Genotyping of Trypanosoma brucei evansi in Egyptian camels: detection of a different non-RoTat 1.2 Trypanosoma brucei evansi in Egyptian camels

Abstract

Trypanosoma brucei evansi (T. b. evansi) is an enzootic organism found in Egyptian camels, which genetically classified into types A and B. To detect the parasite genotype circulating in Egyptian camels, we collected 94 blood samples from three distant districts and subjected them to different PCR assays; T. brucei repeat (TBR), internal transcribed spacer-1 (ITS-1), and variable surface glycoproteins (VSG) (RoTat 1. 2, JN 2118Hu) and EVAB PCRs. The highest prevalence was obtained with TBR (80/91; 87.9%), followed by ITS-1 (52/91; 57.1%), VSG JN 2118Hu (42/91; 46.2%), and VSG RoTat 1. 2 (34/91; 37.4%). We reported a different non-RoTat 1. 2 T. b. evansi for the first time in Egyptian camels. Results showed that 47 (58.7%) out of 80 samples were classified as T. b. evansi. Of these, 14 (29.8%) were RoTat 1. 2 type, 13 (27.6%) were non-RoTat 1. 2 type, and 20 (42.6%) samples were from mixed infection with both types. All samples were tested negative with EVAB PCR. RoTat 1. 2 T. b. evansi was the most prevalent in Giza and El Nubariyah, whereas, in Aswan, the only type detected was non-RoTat 1. 2 T. b. evansi. The nucleotide sequences of the VSG RoTat 1.2 and JN 2118Hu PCR products were submitted to DNA Data Bank of Japan (DDBJ) and GenBank under the accession numbers LC738852, and (OP800400-OP800403). Further research is required to increase the sample size and verify the new sequences to corroborate the prevalence of a new variant of non-RoTat 1.2 T. b. evansi in Egypt.

Keywords: EVAB PCR; Egyptian camels; ITS-1 PCR; JN 2118Hu PCR; Non-RoTat1.2; T. B. evansi; TBR PCR; VSG RoTat 1.2 PCR.

Fig. 1

Map of Egypt shows the locations from which camels blood samples were collected. Samples were collected from small holders in El Nubariyah (20 samples), slaughterhouses in Giza (43), quarantined animals in Aswan (26 samples). The generated map was done using ARcGris online from Esri, Map data © OpenStreetMap contributors, Microsoft, Facebook, and its affilates, Esri community Map contributors, map layer by Esri

Fig. 2

Agarose gel image of DRB-Exon2 PCR products amplified from camel DNA. It shows successful amplification of 457-bp DNA fragment compared with 100-bp DNA ladder in 11 samples out of 14 TBR-negative samples

Fig. 3

Agarose gel image of the PCR products showing detection of T. b. evansi in the camels’ blood. A Amplified products of TBR PCR (164 bp); M, 50-bp ladder; lanes 1–11, positive samples; N, negative control. B Amplified products of ITS-1PCR (90 bp); M, 50-bp ladder; lanes 1–11, positive samples; N, negative control. C Amplified products of RoTat 1.2. PCR (151 bp); M, 50-bp ladder; lanes 1–11, positive samples; N, negative control, and D Amplified products of VSG JN 2118Hu PCR(273 bp); M, 100-bp ladder; lane 1– T. b. evansi type B-positive control; lanes 2–11, some tested samples; 12; RoTat1.2-positive sample; N, negative control

Fig. 4

Agarose gel image of the EVAB PCR products for detection of T. b. evansi type B. It shows negative results of tested samples compared with T. b. evansi type B-positive control (436 bp) and 100-bp DNA ladder

Fig. 5

Agarose gel image of GPI-PLC PCR products of some RoTat1.2 and JN 2118Hu PCR-positive samples for detection of a single copy control gene. It shows amplification of 324-bp DNA fragment of T. b. evansi compared with a 100-bp DNA ladder

Fig. 6

Agarose gel image of GPI-PLC PCR products of RoTat1.2 and JN 2118Hu PCR negative samples for detection of a single copy control gene. It shows amplification of 324-bp DNA fragment in only two samples (one of them is very faint) compared with a 100-bp DNA ladder

Fig. 7

The original phylogenetic tree constructed using 14 partial cd sequences of T. b. evansi RoTat 1.2 and one mRNA complete cd sequence using the Maximum Likelihood method and Jukes–Cantor model (Jukes and Cantor 1969) with 1000 bootstrap replicates conducted in Mega 11 (Tamura et al. 2021). The T. b. evansi RoTat 1.2 type sequences with accession numbers, isolate names, host names, and country where it was isolated, are listed. LC738852 is the accession number of the sequence for our isolate Trypanosoma evansi ARRI VSG gene for variable surface glycoprotein, partial cds indicated by the red square

Fig. 8

Multiple sequence alignment of the sequences of the four sample isolates (OP800400, OP800401, OP800402, OP800403) amplified by VSG JN 2118Hu PCR assay. The sequences were aligned using Clustal W conducted in BioEdit program v7.2.5 (Hall 1999), which indicated that the isolates were identical and sequences were repetitive