Conservation and variation in the region of the Theileria parva p104 antigen coding gene used for PCR surveillance of the parasite

The range of the protozoan parasite Theileria parva, which causes East Coast fever in cattle, has been expanding to countries where it has not previously been detected, as a result of cross-border domestic cattle movement. Countries where T. parva has not previously been observed until recently include Cameroon and South Sudan. This raises the issue of the conservation of the p104 antigen gene, on which the nested PCR assay that is widely used for T. parva surveillance in the blood of infected cattle is based. We sampled 40 isolates from six countries widely distributed across the geographical range of the parasite, including eastern, central and southern Africa, for p104 sequence polymorphism. These included parasites from both domestic cattle and the Cape buffalo (Syncerus caffer) wildlife reservoir. The most frequent allelic variants were present in cattle transmissible isolates from multiple widely separated geographical regions in Zambia, Uganda, Kenya, Tanzania, Rwanda and South Africa. These frequent p104 variants were also present in the three component stocks of the Muguga cocktail used for the infection and treatment live immunisation procedure to control T. parva in the field. Other isolates exhibited unique alleles. This includes some of the p104 sequences from Cameroon, which is outside the known range of the Rhipicephalus tick vector and whose origin is therefore unclear. The nested primer oligonucleotides used to generate the amplicons were universally conserved in cattle-derived parasites and a majority of buffalo-derived isolates across the geographical range of the parasite. However, some rare South African buffalo–derived isolates exhibited one or two mismatches with the primer sequences. It therefore remains possible that some p104 alleles may be so divergent that they do not amplify with the current diagnostic primers and are not detectable in surveys, hence the need for increasing knowledge of genetic heterogeneity of diagnostic targets. There was no evidence for positive selection among those p104 mutations that resulted in residue changes. Importantly, the data indicate that the p104-based PCR detection assay should be effective across the majority of the range of T. parva, and if the one or two mismatches are shown in future to result in the primers annealing less efficiently, then the assay can be further improved by introduction of degenerate bases to enable amplification of the less frequent South African buffalo–derived variant p104 genes

Genetic and antigenic variation of the bovine tick-borne pathogen Theileria parva in the Great Lakes region of Central Africa

BACKGROUND : Theileria parva causes East Coast fever (ECF), one of the most economically important tick-borne diseases of cattle in sub-Saharan Africa. A live immunisation approach using the infection and treatment method (ITM) provides a strong long-term strain-restricted immunity. However, it typically induces a tick-transmissible carrier state in cattle and may lead to spread of antigenically distinct parasites. Thus, understanding the genetic composition of T. parva is needed prior to the use of the ITM vaccine in new areas. This study examined the sequence diversity and the evolutionary and biogeographical dynamics of T. parva within the African Great Lakes region to better understand the epidemiology of ECF and to assure vaccine safety. Genetic analyses were performed using sequences of two antigencoding genes, Tp1 and Tp2, generated among 119 T. parva samples collected from cattle in four agro-ecological zones of DRC and Burundi. RESULTS : The results provided evidence of nucleotide and amino acid polymorphisms in both antigens, resulting in 11 and 10 distinct nucleotide alleles, that predicted 6 and 9 protein variants in Tp1 and Tp2, respectively. Theileria parva samples showed high variation within populations and a moderate biogeographical sub-structuring due to the widespread major genotypes. The diversity was greater in samples from lowlands and midlands areas compared to those from highlands and other African countries. The evolutionary dynamics modelling revealed a signal of selective evolution which was not preferentially detected within the epitope-coding regions, suggesting that the observed polymorphism could be more related to gene flow rather than recent host immune-based selection. Most alleles isolated in the Great Lakes region were closely related to the components of the trivalent Muguga vaccine. CONCLUSIONS : Our findings suggest that the extensive sequence diversity of T. parva and its biogeographical distribution mainly depend on host migration and agro-ecological conditions driving tick population dynamics. Such patterns are likely to contribute to the epidemic and unstable endemic situations of ECF in the region. However, the fact that ubiquitous alleles are genetically similar to the components of the Muguga vaccine together with the limited geographical clustering may justify testing the existing trivalent vaccine for cross-immunity in the region.Additional file 1: Table S1. Cattle blood sample distribution across agroecological zones.Additional file 2: Table S2. Nucleotide and amino acid sequences of Tp1 and Tp2 antigen epitopes from T. parva Muguga reference sequence.Additional file 3: Table S3. Characteristics of 119 T. parva samples obtained from cattle in different agro-ecological zones (AEZs) of The Democratic Republic of Congo and Burundi.Additional file 4: Figure S1. Multiple sequence alignment of the 11 Tp1 gene alleles obtained in this study.Additional file 5: Table S4. Estimates of evolutionary divergence between gene alleles for Tp1 and Tp2, using proportion nucleotide distance.Additional file 6: Table S5. Tp1 and Tp2 genes alleles with their corresponding antigen variants.Additional file 7: Table S6. Amino acid variants of Tp1 and Tp2 CD8+ T cell target epitopes of T. parva from DRC and Burundi.Additional file 8: Figure S2. Multiple sequence alignment of the 10 Tp2 gene alleles obtained in this study.Additional file 9: Table S7. Distribution of Tp1 gene alleles of T. parva from cattle and buffalo in the sub-Saharan region of Africa.Additional file 10: Table S8. Distribution of Tp2 gene alleles of T. parva from cattle and buffalo in the sub-Saharan region of Africa.Additional file 11: Figure S3. Neighbor-joining tree showing phylogenetic relationships among 48 Tp1 gene alleles described in Africa.Additional file 12: Figure S4. Phylogenetic tree showing the relationships among concatenated Tp1 and Tp2 nucleotide sequences of 93 T. parva samples from cattle in DRC and Burundi.This study is part of the PhD work supported by the University of Namur (UNamur, Belgium) through the UNamur-CERUNA institutional PhD grant awarded to GSA for bioinformatic analyses, interpretation of data and manuscript write up in Belgium. The laboratory aspects (molecular biology analysis) of the project were supported by the BecA-ILRI Hub through the Africa Biosciences Challenge Fund (ABCF) programme. The ABCF Programme is funded by the Australian Department for Foreign Affairs and Trade (DFAT) through the BecA-CSIRO partnership; the Syngenta Foundation for Sustainable Agriculture (SFSA); the Bill & Melinda Gates Foundation (BMGF); the UK Department for International Development (DFID); and the Swedish International Development Cooperation Agency (Sida). The ABCF Fellowship awarded to GAS was funded by BMGF grant (OPP1075938). Sample collection, field equipment and preliminary sample processing were supported through the “Theileria” project co-funded to the Université Evangélique en Afrique (UEA) by the Agence Universitaire de la Francophonie (AUF) and the Communauté Economique des Pays des Grands Lacs (CEPGL). The International Foundation for Science (IFS, Stockholm, Sweden) supported the individual scholarship awarded to GSA (grant no. IFS-92890CA3) for field work and part of field equipment to the “Theileria” project.http://www.parasitesandvectors.comam2020Veterinary Tropical Disease

The epidemiology of tick-borne haemoparasites as determined by the reverse line blot hybridization assay in an intensively studied cohort of calves in western Kenya

AbstractThe development of sensitive surveillance technologies using PCR-based detection of microbial DNA, such as the reverse line blot assay, can facilitate the gathering of epidemiological information on tick-borne diseases, which continue to hamper the productivity of livestock in many parts of Africa and elsewhere. We have employed a reverse line blot assay to detect the prevalence of tick-borne parasites in an intensively studied cohort of indigenous calves in western Kenya. The calves were recruited close to birth and monitored for the presence of infectious disease for up to 51 weeks. The final visit samples from 453 calves which survived for the study period were analyzed by RLB. The results indicated high prevalences of Theileria mutans (71.6%), T. velifera (62.8%), Anaplasma sp. Omatjenne (42.7%), A. bovis (39.9%), Theileria sp. (sable) (32.7%), T. parva (12.9%) and T. taurotragi (8.5%), with minor occurrences of eight other haemoparasites. The unexpectedly low prevalence of the pathogenic species Ehrlichia ruminantium was confirmed by a species-specific PCR targeting the pCS20 gene region. Coinfection analyses of the seven most prevalent haemoparasites indicated that they were present as coinfections in over 90% of the cases. The analyses revealed significant associations between several of the Theileria parasites, in particular T. velifera with Theileria sp. sable and T. mutans, and T. parva with T. taurotragi. There was very little coinfection of the two most common Anaplasma species, although they were commonly detected as coinfections with the Theileria parasites. The comparison of reverse line blot and serological results for four haemoparasites (T. parva, T. mutans, A. marginale and B. bigemina) indicated that, except for the mostly benign T. mutans, indigenous cattle seem capable of clearing infections of the three other, pathogenic parasites to below detectable levels. Although the study site was located across four agroecological zones, there was little restriction of the parasites to particular zones

Selection and evaluation of housekeeping genes as endogenous controls for quantification of mRNA transcripts in Theileria parva using quantitative real-time polymerase chain reaction (qPCR).

The reliability of any quantitative real-time polymerase chain reaction (qPCR) experiment can be seriously compromised by variations between samples as well as between PCR runs. This usually result from errors in sample quantification, especially with samples that are obtained from different individuals and tissues and have been collected at various time intervals. Errors also arise from differences in qPCR efficiency between assays performed simultaneously to target multiple genes on the same plate. Consequently, the derived quantitative data for the target genes become distorted. To avoid this grievous error, an endogenous control, with relatively constant transcription levels in the target individual or tissue, is included in the qPCR assay to normalize target gene expression levels in the analysis. Several housekeeping genes (HKGs) have been used as endogenous controls in quantification studies of mRNA transcripts; however, there is no record in the literature of the evaluation of these genes for the tick-borne protozoan parasite, Theileria parva. Importantly, the expression of these genes should be invariable between different T. parva stocks, ideally under different experimental conditions, to gain extensive application in gene expression studies of this parasite. Thus, the expression of several widely used HKGs was evaluated in this study, including the genes encoding β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28S rRNA, cytochrome b and fructose-2.6-biphosphate aldolase (F6P) proteins. The qPCR analysis revealed that the expression of genes encoding cytochrome b, F6P and GAPDH varied considerably between the two T. parva stocks investigated, the cattle-derived T. parva Muguga and the buffalo-derived T. parva 7014. 28S rRNA and β-actin gene expression was the most stable; thus, these genes were considered suitable candidates to be used as endogenous control genes for mRNA quantification studies in T. parva

Analysis of p67 allelic sequences reveals a subtype of allele type 1 unique to buffalo-derived Theileria parva parasites from southern Africa.

East Coast fever (ECF) and Corridor disease (CD) caused by cattle- and buffalo-derived T. parva respectively are the most economically important tick-borne diseases of cattle in the affected African countries. The p67 gene has been evaluated as a recombinant subunit vaccine against ECF, and for discrimination of T. parva parasites causing ECF and Corridor disease. The p67 allele type 1 was first identified in cattle-derived T. parva parasites from East Africa, where parasites possessing this allele type have been associated with ECF. Subsequent characterization of buffalo-derived T. parva parasites from South Africa where ECF was eradicated, revealed the presence of a similar allele type, raising concerns as to whether or not allele type 1 from parasites from the two regions is identical. A 900 bp central fragment of the gene encoding p67 was PCR amplified from T. parva DNA extracted from blood collected from cattle and buffalo in South Africa, Mozambique, Kenya, Tanzania and Uganda, followed by DNA sequence analysis. Four p67 allele types previously described were identified. A subtype of p67 allele type 1 was identified in parasites from clinical cases of CD and buffalo from southern Africa. Notably, p67 allele type 1 sequences from parasites associated with ECF in East Africa and CD in Kenya were identical. Analysis of two p67 B-cell epitopes (TpM12 and AR22.7) revealed amino acid substitutions in allele type 1 from buffalo-derived T. parva parasites from southern Africa. However, both epitopes were conserved in allele type 1 from cattle- and buffalo-derived T. parva parasites from East Africa. These findings reveal detection of a subtype of p67 allele type 1 associated with T. parva parasites transmissible from buffalo to cattle in southern Africa

Analysis of p67 allelic sequences reveals a subtype of allele type 1 unique to buffalo-derived Theileria parva parasites from southern Africa

East Coast fever (ECF) and Corridor disease (CD) caused by cattle- and buffalo-derived T. parva respectively are the most economically important tick-borne diseases of cattle in the affected African countries. The p67 gene has been evaluated as a recombinant subunit vaccine against ECF, and for discrimination of T. parva parasites causing ECF and Corridor disease. The p67 allele type 1 was first identified in cattle-derived T. parva parasites from East Africa, where parasites possessing this allele type have been associated with ECF. Subsequent characterization of buffalo-derived T. parva parasites from South Africa where ECF was eradicated, revealed the presence of a similar allele type, raising concerns as to whether or not allele type 1 from parasites from the two regions is identical. A 900 bp central fragment of the gene encoding p67 was PCR amplified from T. parva DNA extracted from blood collected from cattle and buffalo in South Africa, Mozambique, Kenya, Tanzania and Uganda, followed by DNA sequence analysis. Four p67 allele types previously described were identified. A subtype of p67 allele type 1 was identified in parasites from clinical cases of CD and buffalo from southern Africa. Notably, p67 allele type 1 sequences from parasites associated with ECF in East Africa and CD in Kenya were identical. Analysis of two p67 Bcell epitopes (TpM12 and AR22.7) revealed amino acid substitutions in allele type 1 from buffalo-derived T. parva parasites from southern Africa. However, both epitopes were conserved in allele type 1 from cattle- and buffalo-derived T. parva parasites from East Africa. These findings reveal detection of a subtype of p67 allele type 1 associated with T. parva parasites transmissible from buffalo to cattle in southern Africa.S1 Fig. PCR amplicons from cattle- and buffalo-derived T. parva parasites from East and southern Africa. (a) p67 PCR amplicons from buffalo-derived T. parva parasites from clinical cases of Corridor disease (CD) and non-clinical T. parva-positive cases from South Africa (SA); (b) p67 PCR amplicons from buffalo-derived T. parva parasites originating from buffalo in Hluhluwe-iMfolozi Park, KwaZulu-Natal; (c) p67 PCR amplicons from cattle-derived T. parva parasites originating from cattle in Mbarara in Western Uganda. 1kb DNA ladder (#SM0311, ThermoFisher Scientific, Waltham, MA USA) was used in (a), 100bp plus DNA ladder (#SM0321, ThermoFisher Scientific, Waltham, MA USA) was used in (b) and (c). SA— South Africa; CD—Corridor disease; M—molecular weight marker.S1 Table. The Ct values of samples from active clinical cases of Corridor disease and nonclinical T. parva-positive cases collected from Mpumalanga province in South Africa.S2 Table. Predicted protein sequence alignment of allele type 2 identified in T. parva parasites from cattle and buffalo.S3 Table. Predicted protein sequence alignment of allele type 3 identified in T. parva parasites from cattle and buffalo.S4 Table. Predicted protein sequence alignment of allele type 4 identified in T. parva parasites from cattle and buffalo.S5 Table. Estimates of the evolutionary divergence between sequences of allele type 1 from T. parva parasites from East and Southern Africa.S6 Table. Taxonomic metadata detailing the grouping of p67 allele types from T. parva parasites from East and Southern Africa.The National Research Foundation, South Africa, the University of Pretoria, South Africa, the National Research Fund, Kenya and the KPS - NRF, South Africa.http://www.plosone.orgam2021Veterinary Tropical Disease

Genetic analysis of the VP2-encoding gene of canine parvovirus strains from Africa

Since the emergence of canine parvovirus type-2 (CPV-2) in the early 1970s, it has been evolving into novel genetic and antigenic variants (CPV-2a, 2b and 2c) that are unevenly distributed throughout the world. Genetic characterization of CPV-2 has not been documented in Africa since 1998 apart from the study carried out in Tunisia 2009. A total of 139 field samples were collected from South Africa and Nigeria, detected using PCR and the full length VP2-encoding gene of 27 positive samples were sequenced and genetically analyzed. Nigerian samples (n = 6), South Africa (n = 19) and vaccine strains (n = 2) were compared with existing sequences obtained from GenBank. The results showed the presence of both CPV-2a and 2b in South Africa and only CPV-2a in Nigeria. No CPV-2c strain was detected during this study. Phylogenetic analysis showed a clustering not strictly associated with the geographical origin of the analyzed strains, although most of the South African strains tended to cluster together and the viral strains analyzed in this study were not completely distinct from CPV-2 strains from other parts of the world. Amino acid analysis showed predicted amino acid changes. (C) 2013 Elsevier B.V. All rights reserved

Gene expression stability of five candidate <i>T</i>. <i>parva</i> reference genes as assessed by RefFinder.

<p>Gene expression stability of five candidate <i>T</i>. <i>parva</i> reference genes as assessed by RefFinder.</p

Specificity of real-time PCR amplification: Melting curves generated after amplification of five candidate <i>T</i>. <i>parva</i> reference genes showing a single melting peak for each product.

<p>Each experiment included two biological replicates of cDNA prepared from RNA isolated from cell cultures infected with <i>T</i>. <i>parva</i> Muguga and <i>T</i>. <i>parva</i> 7014 and a no template control (NTC).</p

Student’s <i>t</i>-test results for analysis of intra-assay variations in the expression of HKGs between <i>T</i>. <i>parva</i> Muguga and 7014.

<p>The results from the first run are presented in panel A and results from the second run in panel B.</p