Assessment of sequence descriptions of selected Theileria parva hypothetical proteins retrieved from sequence similarity search databases

The protozoan parasite Theileria parva is the causative agent of cattle theileriosis, a disease with a destructive impact on the agricultural economy through mortality and morbidity of affected cattle. In cattle, T. parva infection results in varied disease syndromes depending on the parasite host of origin; cattle-derived T. parva causes East Coast fever while buffaloderived parasites cause Corridor disease. The differences in the resulting disease caused by T. parva infection have raised an interest to understand the proteins involved in the disease manifestations. Consequently, a transcriptome study comparing the cattle and buffalo-derived T. parva isolates was undertaken; differentially expressed genes were detected of which 74% (867) were hypothetical proteins (HPs). Since HPs could play a vital role in the pathogenicity and host-parasite interaction, the primary aim of the study was to identify biological roles of these proteins. A combination of in silico analysis tools was employed to annotate HPs according to sequence descriptions, confirmed by sequence homology in comparison with closely related species and conserved domains. Initial screening for sequence descriptions (SDs) based on sequence similarity search using Blast2GO retrieved results for 392 HPs. Comparison of this output to other databases (KEGG and KOBAS) detected consensus SDs for 229 HPs, of which 109 were further confirmed by inferring homology to related species. Sequence homology analysis also resulted in designation of SDs to 74 HPs from the remaining 163 without consensus SDs from database analyses. For HPs which did not meet the criteria employed in sequence homology analysis (209), conserved domain analysis facilitated assigning of SDs for 114 HPs. Overall, 297 (76%) HPs were successfully allocated SDs. Finally, the results from this study have showed that output from automated sequence similarity databases is not always reliable in assigning SDs for specific species, making confirmation using other approaches necessary.Poster presented at the University of Pretoria, Faculty of Veterinary Science Faculty Day, August 25, 2016, Pretoria, South Africa.ab201

Molecular analysis of South African ovine herpesvirus 2 strains based on selected glycoprotein and tegument genes

Ovine herpesvirus 2 (OvHV-2), is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a generally fatal disease of cattle and other captive wild ruminants. Information on the OvHV-2 strains circulating in South Africa (SA) and other African countries with regard to genetic structure and diversity, and pattern of distribution is not available. This study aimed to characterize the OvHV-2 strains circulating in SA using selected genes encoding glycoproteins and tegument proteins. To establish the genetic diversity of OvHV-2 strains, four genes, Ov 7, Ov 8 ex2, ORF 27 and ORF 73 were selected for analysis by PCR and DNA sequencing. Nucleotide and amino acid multiple sequence analyses revealed two genotypes for ORF 27 and ORF 73, and three genotypes for Ov 7 and Ov 8 ex2, randomly distributed throughout the regions. Ov 7 and ORF 27 nucleotide sequence analysis revealed variations that distinguished SA genotypes from those of reference OvHV-2 strains. Epitope mapping analysis showed that mutations identified from the investigated genes are not likely to affect the functions of the gene products, particularly those responsible for antibody binding activities associated with B-cell epitopes. Knowledge of the extent of genetic diversity existing among OvHV-2 strains has provided an understanding on the distribution patterns of OvHV-2 strains or genotypes across the regions of South Africa. This can facilitate the management of SA-MCF in SA, in terms of introduction of control measures or safe practices to monitor and control OvHV-2 infection. The products encoded by the Ov 7, Ov 8 ex2 and ORF 27 genes are recommended for evaluation of their coded proteins as possible antigens in the development of an OvHV-2 specific serodiagnostic assay.S1 Table. Average sequence identities determined for the Ov 7 nucleotide and amino sequences obtained between South African Ov 7 sequences compared to reference sequences.S2 Table. Average sequence identities determined for the Ov 8 ex2 nucleotide (Figure A) and derived amino acid (Figure B) sequences obtained between South African OvHV-2 strains compared to reference strains.S3 Table. Average sequence identities determined for the ORF 27 nucleotide and derived amino acid sequences obtained between South African OvHV-2 strains compared to reference strains.S4 Table. Average sequence identities for the ORF 73 nucleotide and derived amino acid sequences obtained between South African OvHV-2 strains compared to reference strains.This work was supported by Department of Science and Technology (DST) for research funding; Meat Industry Trust (MIT) for payment of the University tuitions.http://www.plosone.orgam2017Veterinary Tropical Disease

Molecular analysis of South African ovine herpesvirus 2 strains based on selected glycoprotein and tegument genes

Ovine herpesvirus 2 (OvHV-2), is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a generally fatal disease of cattle and other captive wild ruminants. Information on the OvHV-2 strains circulating in South Africa (SA) and other African countries with regard to genetic structure and diversity, and pattern of distribution is not available. This study aimed to characterize the OvHV-2 strains circulating in SA using selected genes encoding glycoproteins and tegument proteins. To establish the genetic diversity of OvHV-2 strains, four genes, Ov 7, Ov 8 ex2, ORF 27 and ORF 73 were selected for analysis by PCR and DNA sequencing. Nucleotide and amino acid multiple sequence analyses revealed two genotypes for ORF 27 and ORF 73, and three genotypes for Ov 7 and Ov 8 ex2, randomly distributed throughout the regions. Ov 7 and ORF 27 nucleotide sequence analysis revealed variations that distinguished SA genotypes from those of reference OvHV-2 strains. Epitope mapping analysis showed that mutations identified from the investigated genes are not likely to affect the functions of the gene products, particularly those responsible for antibody binding activities associated with B-cell epitopes. Knowledge of the extent of genetic diversity existing among OvHV-2 strains has provided an understanding on the distribution patterns of OvHV-2 strains or genotypes across the regions of South Africa. This can facilitate the management of SA-MCF in SA, in terms of introduction of control measures or safe practices to monitor and control OvHV-2 infection. The products encoded by the Ov 7, Ov 8 ex2 and ORF 27 genes are recommended for evaluation of their coded proteins as possible antigens in the development of an OvHV-2 specific serodiagnostic assay.S1 Table. Average sequence identities determined for the Ov 7 nucleotide and amino sequences obtained between South African Ov 7 sequences compared to reference sequences.S2 Table. Average sequence identities determined for the Ov 8 ex2 nucleotide (Figure A) and derived amino acid (Figure B) sequences obtained between South African OvHV-2 strains compared to reference strains.S3 Table. Average sequence identities determined for the ORF 27 nucleotide and derived amino acid sequences obtained between South African OvHV-2 strains compared to reference strains.S4 Table. Average sequence identities for the ORF 73 nucleotide and derived amino acid sequences obtained between South African OvHV-2 strains compared to reference strains.This work was supported by Department of Science and Technology (DST) for research funding; Meat Industry Trust (MIT) for payment of the University tuitions.http://www.plosone.orgam2017Veterinary Tropical Disease

Limited diversity in the CD8+ antigen-coding loci in Theileria parva parasites from cattle from southern and eastern Africa

Theileria parva infections in cattle causes huge economic losses in the affected African countries, directly impacting the livelihood of the poor small-holder farmers. The current immunization protocol using live sporozoites in eastern Africa, is among the control measures designed to limit T. parva infections in cattle. However, the ability of the immune protection induced by this immunization to protect against field parasites has been compromised by the diversity of the parasite involving the schizont antigen genes. Previous studies have reported on the antigenic diversity of T. parva parasites from southern and eastern Africa, however, similar reports on T. parva parasites particularly from cattle from southern Africa remains scanty, due to the self-limiting nature of Corridor disease. Thus, we evaluated the diversity of CD8+ T-cell regions of ten schizont antigen genes in T. parva parasites associated with Corridor disease and East Coast fever (ECF) from southern and eastern Africa respectively. Regions of schizont antigen (TpAg) genes containing the CD8+ T-cell epitopes (CTL determinants) were amplified from genomic DNA extracted from blood of T. parva positive samples, cloned and sequenced. The results revealed limited diversity between the two parasite groups from cattle from southern and eastern Africa, defying the widely accepted notion that antigen-encoding loci in cattle-derived parasites are conserved, while in buffalo-derived parasites, they are extensively variable. This suggests that only a sub-population of parasites is successfully transmitted from buffalo to cattle, resulting in the limited antigenic diversity in Corridor disease parasites. Tp4, Tp5, Tp7 and Tp8 showed limited to absence of diversity in both parasite groups, suggesting the need to further investigate their immunogenic properties for consideration as candidates for a subunit vaccine. Distinct and common variants of Tp2 were detected among the ECF parasites from eastern Africa indicating evidence of parasite mixing following immunization. This study provides additional information on the comparative diversity of TpAg genes in buffalo- and cattle-derived T. parva parasites from cattle from southern and eastern Africa.The National Research Foundation, South Africa; University of Pretoria, South Africa; and the National Research Fund, Kenya.https://www.elsevier.com/locate/vetparhj2022Veterinary Tropical Disease

Molecular characterization of Theileria parva field strains derived from cattle and buffalo sympatric populations of Northern Tanzania

East coast fever is a disease of cattle caused by Theileria parva and is transmitted by the tick Rhipicephalus appendiculatus. Buffalos are a natural reservoir of the parasite. The objectives of this study were to determine the T. parva infection in buffalo and cattle field samples, to identify genotypes of T. parva infecting cattle and buffalo and finally to investigate the presence of buffalo-derived T. parva genotypes in cattle that graze in close proximity with infected buffalo. Real-Time PCR (RT-PCR) and Fluorescence Resonance Energy Transfer (FRET) was used to diagnose T.parva. Genotyping of T. parva was conducted by analysing p67, PIM and p104 genes . In addition, amplicons with unique PIM and p104 PCR-RFLP profiles were sequenced to identify shared genotypes. The results have shown the high occurrence of T. parva in buffalo and a much higher occurrence of T. parva in cattle from the Ngorongoro area. Analysis of the variable region of the p67 loci showed four different T. parva alleles in buffalo and cattle. PCR-RFLP analysis of the p104 loci revealed four allelic profiles in buffalo and two in cattle. PCR-RFLP analysis of PIM loci from buffalo gave profiles that were complex and difficult to interpret. On the contrary, cattle showed allelic profiles that resemble Muguga and Marikebuni isolate PIM gene profiles. Multi-locus genotypes (MLGs) and sequence analysis revealed that the majority of buffalo and cattle genotypes were distinct but there is an indication of some sharing of genotypes between buffalo and cattle.Belgian Directorate General for Development Co-operation Framework agreement ITM/DGCD and the Institute of Tropical Medicine (ITM).http://www.usa-journals.comam201

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 cattlederived 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.This work was funded by the Genomics Research Institute of the University of Pretoria, (http://www.up.ac.za/the-genomics-research- institute/home) to KPS-M.http://www.plosone.orgam2018Veterinary Tropical Disease

Molecular detection of zoonotic rickettsiae and Anaplasma spp. in domestic dogs and their ectoparasites in Bushbuckridge, South Africa

Members of the order Rickettsiales are small, obligate intracellular bacteria that are vector-borne and can cause mild to fatal diseases in humans worldwide. There is little information on the zoonotic rickettsial pathogens that may be harbored by dogs from rural localities in South Africa. To characterize rickettsial pathogens infecting dogs, we screened 141 blood samples, 103 ticks, and 43 fleas collected from domestic dogs in Bushbuckridge Municipality, Mpumalanga Province of South Africa, between October 2011 and May 2012 using the reverse line blot (RLB) and Rickettsia genus and species-specific quantitative real-time PCR (qPCR) assays. Results from RLB showed that 49% of blood samples and 30% of tick pools were positive for the genus-specific probes for Ehrlichia/Anaplasma; 16% of the blood samples were positive for Ehrlichia canis. Hemoparasite DNA could not be detected in 36% of blood samples and 30% of tick pools screened. Seven (70%) tick pools and both flea pools were positive for Rickettsia spp; three (30%) tick pools were positive for Rickettsia africae; and both flea pools (100%) were positive for Rickettsia felis. Sequencing confirmed infection with R. africae and Candidatus Rickettsia asemboensis; an R. felis-like organism from one of the R. felis-positive flea pools. Anaplasma sp. South Africa dog strain (closely related to Anaplasma phagocytophilum), A. phagocytophilum, and an Orientia tsutsugamushi-like sequence were identified from blood samples. The detection of emerging zoonotic agents from domestic dogs and their ectoparasites in a rural community in South Africa highlights the potential risk of human infection that may occur with these pathogens.The HDSS-Dogs platform (protocol no. VO33-11) was supported by funding to Darryn Knobel from the Morris Animal Foundation, United States (grant no.D12CA-312). Drs. A.N. Maina and A.L. Richards were supported by funding of the Global Emerging Infections Surveillance and Response System, work unit # A1402.http://online.liebertpub.com/VBZ2017-04-30hb2016Veterinary Tropical Disease

Occurrence of tick-borne haemoparasites in cattle in the Mungwi District, Northern Province, Zambia

Little is known about the occurrence of haemoparasites in cattle in communal grazing areas of Mungwi District of Northern Province, Zambia. Clinical signs and post mortem lesions are pathognomonic of mixed tick-borne infections especially babesiosis, anaplasmosis and East Coast fever. The main objective of this study was to screen selected communal herds of cattle for tick-borne haemoparasites, and identify the tick vectors associated with the high cattle mortalities due to suspected tick-borne diseases in the local breeds of cattle grazing along the banks of the Chambeshi River in Mungwi District, Northern Province, Zambia. A total of 299 cattle blood samples were collected from July to September 2010 from Kapamba (n = 50), Chifulo (n = 102), Chisanga (n = 38), Kowa (n = 95) and Mungwi central (n = 14) in the Mungwi District. A total of 5288 ticks were also collected from the sampled cattle from April to July 2011. DNA was extracted from the cattle blood and the hypervariable region of the parasite small subunit rRNA gene was amplified and subjected to the reverse line blot (RLB) hybridization assay. The results of the RLB assay revealed the presence of tick-borne haemoparasites in 259 (86.6%) cattle blood samples occurring either as single (11.0%) or mixed (75.6%) infections. The most prevalent species present were the benign Theileria mutans (54.5%) and T. velifera (51.5%). Anaplasma marginale (25.7%), Babesia bovis (7.7%) and B. bigemina (3.3%) DNA were also detected in the samples. Only one sample (from Kapamba) tested positive for the presence of T. parva. This was an unexpected finding; also because the tick vector, Rhipicephalus appendiculatus, was identified on animals from Kowa (14.0%), Chisanga (8.5%), Chifulo (6.0%) and Kapamba (1.4%). One sample (from Kapamba) tested positive for the presence of Ehrlichia ruminantium even though Amblyomma variegatum ticks were identified from 52.9% of the sampled animals from all study areas. There was significant positive association between T. mutans and T. velifera (p < 0.001) infections, and between A. marginale and B. bovis (p = 0.005). The presence of R. microplus tick vectors on cattle was significantly associated with B. bovis (odds ratio, OR = 28.4, p < 0.001) and A. marginale (OR = 42.0, p < 0.001) infections, while A. variegatum presence was significantly associated with T. mutans (OR = 213.0, p < 0.001) and T. velifera (OR = 459.0, p < 0.001) infections. Rhipicephalus decoloratus was significantly associated with B. bigemina (OR = 21.6, p = 0.004) and A. marginale (OR = 28.5, p < 0.001). Multivariable analysis showed a significant association between location and tick-borne pathogen status for A. marginale (p < 0.001), T. mutans (p = 0.004), T. velifera (p = 0.003) and T. taurotragi (p = 0.005). The results of our study suggest that the cause of cattle mortalities in Mungwi during the winter outbreaks is mainly due to A. marginale, B. bovis and B. bigemina infections. This was confirmed by the clinical manifestation of the disease in the affected cattle and the tick species identified on the animals. The relatively low prevalence of T. parva, B. bigemina, B. bovis and E. ruminantium could indicate the existence of endemic instability with a pool of susceptible cattle and the occurrence of disease outbreaks.The Belgian Directorate General for Development Co-operation Framework agreement ITM/DGCD and the South African National Research Foundation (NRF) (Grant 76529 to Marinda Oosthuizen).http://www.elsevier.com/locate/ttbdis2019-03-01hj2018Veterinary Tropical Disease

Molecular detection and characterisation of protozoan and rickettsial pathogens in ticks from cattle in the pastoral area of Karamoja, Uganda

Ticks and tick-borne diseases (TBDs) significantly affect cattle production and the livelihoods of communities in pastoralist areas. Data on protozoan and rickettsial pathogens in ticks infesting cattle in Uganda is scanty; while it is an indicator of the likelihood of disease transmission and occurrence. A cross-sectional study was conducted amongst cattle in the Karamoja Region, northeastern Uganda, from July through September 2017, to determine the tick species diversity, identify protozoan and rickettsial pathogens in the ticks, and characterise pathogenic species by sequence and phylogenetic analyses. About 50 % of the ticks detected from each predilection site on each animal were collected from 100 purposively-selected cattle from 20 randomly-selected herds. Twelve tick species belonging to the genera Amblyomma, Rhipicephalus and Hyalomma were identified, the most abundant being Amblyomma lepidum (93.9 %), followed by Amblyomma variegatum (2.0 %) and Rhipicephalus evertsi evertsi (1.0 %). Tick species that have not been reported in recent studies amongst cattle in Uganda were found, namely Rhipicephalus pravus, Rhipicephalus praetextatus and Rhipicephalus turanicus. The ticks were grouped into 40 pools, by species and location, and the reverse line blot (RLB) hybridisation assay was used to detect pathogens from the ticks. The most frequently detected tick-borne parasites were Theileria mutans, Theileria velifera and Theileria parva, each observed in 25 % (10/40) of the tick pools. Tick-borne pathogens, namely Babesia rossi, Babesia microti and Theileria sp. (sable) that are not common to, or not known to infect, cattle were identified from ticks. The gene encoding Ehrlichia ruminantium pCS20 region, the Ehrlichia and Anaplasma 16S rRNA gene, and T. parva p67 sporozoite antigen gene were amplified, cloned and sequenced. Seven novel E. ruminantium pCS20 variants were identified, and these grouped into two separate clusters with sequences from other parts of Africa and Asia. The T. parva p67 sequences were of the allele type 1, and parasites possessing this allele type are commonly associated with East Coast fever in eastern Africa. Analysis of the Ehrlichia and Anaplasma 16S rRNA gene sequences showed that they were closely related to Rickettsia africae and to a new Ehrlichia species variant recently found in China. Our R. africae 16S rRNA sequences grouped with R. africae isolates from Nigeria, Egypt and Benin. The information on tick species diversity and pathogens in the various tick species provides an indicator of potential transmission amongst cattle populations, and to humans, and can be useful to estimate disease risk and in control strategies.The Directorate-General for Development Cooperation (DGDC) through the Institute of Tropical Medicine (ITM) in Antwerp, Belgium (Flanders), the University of Pretoria, South Africa and the Claude Leon Foundation, South Africa.https://www.elsevier.com/locate/ttbdishj2022Veterinary Tropical Disease

Tick-borne haemoparasites in African buffalo (Syncerus caffer) from two wildlife areas in northern Botswana

BACKGROUND : The African buffalo (Syncerus caffer) is a host for many pathogens known to cause economically important diseases and is often considered an important reservoir for livestock diseases. Theileriosis, heartwater, babesiosis and anaplasmosis are considered the most important tick-borne diseases of livestock in sub-Saharan Africa, resulting in extensive economic losses to livestock farmers in endemic areas. Information on the distribution of tick-borne diseases and ticks is scarce in Northern Botswana. Nevertheless, this data is necessary for targeting surveillance and control measures in livestock production at national level. METHODS : In order to address this gap, we analyzed 120 blood samples from buffalo herds for the presence of common tick-borne haemoparasites causing disease in livestock, collected in two of the main wildlife areas of Northern Botswana: the Chobe National Park (CNP, n = 64) and the Okavango Delta (OD, n = 56). RESULTS : Analysis of the reverse line blot (RLB) hybridization assay results revealed the presence of Theileria, Babesia, Anaplasma and Ehrlichia species, either as single or mixed infections. Among the Theileria spp. present, T. parva (60%) and T. mutans (37%) were the most prevalent. Other species of interest were Anaplasma marginale subsp. centrale (30%), A. marginale (20%), Babesia occultans (23%) and Ehrlichia ruminantium (6%). The indirect fluorescent antibody test (IFAT) indicated 74% of samples to be positive for the presence of T. parva antibodies. Quantitative real-time PCR (qPCR) detected the highest level of animals infected with T. parva (81% of the samples). The level of agreement between the tests for detection of T. parva positive animals was higher between qPCR and IFAT (kappa = 0.56), than between qPCR and RLB (kappa = 0.26) or the latter and IFAT (kappa = 0.15). CONCLUSIONS : This is the first report of tick-borne haemoparasites in African buffalo from northern Botswana, where animals from the CNP showed higher levels of infection than those from OD. Considering the absence of fences separating wildlife and livestock in the CNP and the higher levels of some parasite species in buffalo from that area, surveillance of tick-borne diseases in livestock at the interface in the CNP should be prioritized.United Nations Food and Agriculture Organization ECTAD Office in Gaborone (LoAPR 43231, New PR 45371) and the South African National Research Foundation (NRF, CSUR program: SUR2009062200001347). It also falls under the Belgian Directorate General for Development Co-operation Framework agreement ITM/DGCD.http://www.parasitesandvectors.comhb201