Diversity of Theileria parasites in African buffalo (Syncerus caffer) and the challenge of differential diagnosis

In South Africa, the diagnosis of Theileria parva in cattle and buffalo has been complicated by the presence of mildly pathogenic and non-pathogenic Theileria spp. This can lead to inaccurate diagnostic results and confuse the epidemiology of theileriosis. The aims of this study were to identify and characterize the 18S rRNA genes of novel Theileria spp. of the African buffalo, as well as to test new gene targets that will allow for the development of more accurate diagnostic tests for the identification of T. parvainfections in cattle and buffalo. Buffalo blood samples originating from different geographical regions in South Africa and from Mozambique were screened for the presence of Theileria spp. by the reverse line blot (RLB) hybridization assay. A total of six Theileria spp., namely T. parva, Theileria sp. (buffalo), Theileria mutans, Theileria velifera and Theileria buffeli, were identified from the buffalo samples. These occurred mainly as mixed infections. Some of the samples hybridized only with the Theileria/Babesia genus specific probe that is used in the RLB assay, and not with any of the species-specific probes used, suggesting the presence of novel genotypes or species. The full-length 18S rRNA genes of parasites from selected samples were characterized by cloning and sequencing. In addition to the identification of 18S rRNA gene sequences that were similar to published Theileria spp. of cattle and buffalo, we identified Theileria sp. (bougasvlei), and novel 18S rRNA gene variants of T. mutans, T. velifera, T. bufJeli. This variation explained why the RLB hybridization assay failed to detect these species in some of the analysed samples. As extensive variation was observed within the T. mutan group, specific RLB oligonucleotide probes were designed from the V 4 hypervariable region of the T. mutans-like 1 and 2/3 18S rRNA gene sequences. Unfortunately these cross-hybridized with T. mutans target DNA and could not be used to screen buffalo samples to determine the occurrence of these genotypes in buffalo in South Africa. This problem could be solved by designing probes from a more variable area of the 18S rRNA gene of the T. mutans groups. Alternatively, a quantitative real-time PCR (qPCR) assay could be used for differentiation of these genotypes as it is more sensitive than the RLB assay. Despite the variation observed in the full-length T parva 18S rRNA gene sequences, the area in the V 4 hypervariable region where the T parva RLB and real-time PCR hybridization probes were developed was relatively conserved between sequences obtained in this study. The existing T parva-specific qPCR assay was able to successfully detect all T parva variants identified in this study and, although amplicons were obtained from Theileria sp. (buffalo) and Theileria sp. (bougasvlei) DNA, these species were not detected by the T parva-specific hybridization probes. The sequences of the other Theileria spp. and the novel genotypes identified in this study under the probes were also different from that of T parva and therefore these species do not compromise the specificity of the T parva 18S qPCR assay. In order to determine the sequence variation and phylogenetic positions of T buffeli spp. of the African buffalo, we cloned and sequenced their 18S rRNA gene and complete internal transcribed spacer (ITS). We identified novel T buffeli-like and T sinensis-like 18S rRNA and ITS genotypes from buffalo originating from two different geographical regions in South Africa. There was extensive sequence variation between these novel South African genotypes and known T buffeli-like and T sinensis-like genotypes. The presence of organisms with T buffeli-like and T. sinensis-like genotypes in the African buffalo is of significant importance, particularly to the cattle industry in South Africa as these animals might act as sources of infections to naIve cattle. Recently, a qPCR assay based on the cox III gene was developed for the diagnosis of Theileria spp. in cattle. This test detects and differentiates six Theileria spp. in cattle. We evaluated the use of this assay for the detection of Theileria spp. in buffalo. The results of the cox III qPCR were compared to those of the RLB and 18S qPCR for the simultaneous detection and differentiation of Theileria spp. of the African buffalo, and for the specific detection of T parva, respectively. The cox III genes from selected samples with non-specific melting peaks were characterized by cloning and sequencing. Extensive sequence variation in the cox III gene was observed between and within species. The T mutans group was the most variable. The qPCR assay could be further improved by designing new primers and probes using all known cox III gene sequences of Theileria spp. Of buffalo and cattle. This study highlights the complexity of the diagnosis of T parva in cattle and buffalo in South Africa. It provides invaluable information towards the development of an improved molecular diagnostic assay for T parva and co-infecting species in cattle and buffalo in South Africa which will assist the veterinary regulatory authorities in the control of Corridor disease in South Africa.Thesis (PhD)--University of Pretoria, 2011.Veterinary Tropical DiseasesUnrestricte

Phylogeny of Theileria buffeli genotypes identified in the South African buffalo (Syncerus caffer) population

Theileria buffeli/orientalis is a group of benign and mildly pathogenic species of cattle andbuffalo in various parts of the world. In a previous study, we identified T. buffeli in blood sam-ples originating from the African buffalo (Syncerus caffer) in the Hluhluwe–iMfolozi GamePark (HIP) and the Addo Elephant Game Park (AEGP) in South Africa. The aim of this studywas to characterise the 18S rRNA gene and complete internal transcribed spacer (ITS1-5.8S-ITS2) region of T. buffeli samples, and to establish the phylogenetic position of this speciesbased on these loci. The 18S rRNA gene and the complete ITS region were amplified fromDNA extracted from blood samples originating from buffalo in HIP and AEGP. The PCR prod-ucts were cloned and the resulting recombinants sequenced. We identified novel T. buffeli-like 18S rRNA and ITS genotypes from buffalo in the AEGP, and novel Theileria sinensis-like18S rRNA genotypes from buffalo in the HIP. Phylogenetic analyses indicated that the T.buffeli-like sequences were similar to T. buffeli sequences from cattle and buffalo in Chinaand India, and the T. sinensis-like sequences were similar to T. sinensis 18S rRNA sequencesof cattle and yak in China. There was extensive sequence variation between the novel T.buffeli genotypes of the African buffalo and previously described T. buffeli and T. sinensisgenotypes. The presence of organisms with T. buffeli-like and T. sinensis-like genotypes inthe African buffalo could be of significant importance, particularly to the cattle industry inSouth Africa as these animals might act as sources of infections to naïve cattle. This is thefirst report on the characterisation of the full-length 18S rRNA gene and ITS region of T.buffeli and T. sinensis genotypes in South Africa. Our study provides invaluable informationtowards the classification of this complex group of benign and mildly pathogenic species.South African National Research Foundation (NRFICD2006072000009) and UP Research Development Programme.http://www.elsevier.com/locate/vetparhb201

Sequence variation identified in the 18S rRNA gene of Theileria mutans and Theileria velifera from the African buffalo (Syncerus caffer)

The African buffalo (Syncerus caffer) is a natural reservoir host for both pathogenic and nonpathogenic Theileria species. These often occur naturally as mixed infections in buffalo. Although the benign and mildly pathogenic forms do not have any significant economic importance, their presence could complicate the interpretation of diagnostic test results aimed at the specific diagnosis of the pathogenic T. parva in cattle and buffalo in South Africa. The 18S rRNA gene has been used as the target in a quantitative real-time PCR (qPCR) assay for the detection of T. parva infections. However, the extent of sequence variation within this gene in the non-pathogenic Theileria spp. of the Africa buffalo is not well known. The aim of this study was, therefore, to characterize the full-length 18S rRNA genes of T. mutans, Theileria sp. (strain MSD) and T. velifera and to determine the possible influence of any sequence variation on the specific detection of T. parva using the 18S rRNA qPCR. The reverse line blot (RLB) hybridization assay was used to select samples which either tested positive for several different Theileria spp., or which hybridized only with the Babesia/Theileria genus-specific probe and not with any of the Babesia or Theileria speciesspecific probes. The full-length 18S rRNA genes from 14 samples, originating from 13 buffalo and one bovine from different localities in South Africa, were amplified, cloned and the resulting recombinants sequenced. Variations in the 18S rRNA gene sequences were identified in T. mutans, Theileria sp. (strain MSD) and T. velifera, with the greatest diversity observed amongst the T. mutans variants. This variation possibly explained why the RLB hybridization assay failed to detect T. mutans and T. velifera in some of the analysed samples.South African National Research Foundation (NRF ICD2006072000009) and UP Research Development Programme.http://www.elsevier.com/locate/vetparhb2013ab201

Distribution and prevalence of ticks and tick-borne pathogens of wild animals in South Africa : a systematic review

Ticks are significant ectoparasites of animals and humans. Published data indicate that most vectors that transmit livestock and human pathogens in sub-Saharan Africa, are native to the region and originate from wild animals. Currently, there is a paucity of information on the role of wild animals on the epidemiology of zoonotic tick-borne pathogens in South Africa. This systematic review focuses on the distribution of ticks and prevalence of tick-borne pathogens in different wild animals in South Africa to identify potential reservoir hosts and possible hotspots for emergence of novel tick-borne pathogens. Following several screening processes, 38 peer-reviewed studies published from 1970 to 2021, were deemed eligible. The studies reported on ticks collected from 63 host species of 21 host families, mostly Canidae, Felidae, Bovidae and Muridae. A total of 49 tick species of nine genera, i.e. Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, Margaropus, Nuttalliella, Rhipicentor and Rhipicephalus, were reported. Nine tick species, i.e. Amblyomma marmoreum, Am. hebraeum, Haemaphysalis elliptica, Hyalomma truncatum, I. rubicundus, Rh. appendiculatus, Rh. (B.) decoloratus, Rh. evertsi evertsi and Rh. simus were the most commonly reported. Pathogens of the genera Anaplasma, Babesia, Hepatozoon and Theileria were identified in the wild animals. This review provides more insight on the ecology of ticks and tick-borne pathogens of wild animals in South Africa and gives useful information for predicting their future spread. It also demonstrates that wild animals habour a diverse range of tick species. This level of diversity entails a similarly high potential for emergence of novel tick-borne pathogens. The review further indicates that wild animals in South Africa are sentinels of tick-borne protozoans of veterinary importance and some bacterial pathogens as most ticks they habour are known vectors of pathogens of domestic animals and humans. However, studies on potential tick-borne zoonoses are under-represented and should be included in future epidemiological surveys, especially in the light of climate change and other anthropogenic threats which might result in the emergence of novel tick-borne pathogens.South African National Biodiversity Institute (SANBI) and the National Research Foundation (NRF) of South Africa.https://www.sciencedirect.com/journal/current-research-in-parasitology-and-vector-borne-diseaseshj2023Veterinary Tropical Disease

Molecular detection and phylogenetic analysis of Anaplasma marginale and Anaplasma centrale amongst transhumant cattle in north-eastern Uganda

There is little molecular data from Anaplasma marginale and Anaplasma centrale isolates from cattle in Uganda. Between November 2013 and January 2014, blood was collected from 240 cattle in 20 randomly-selected herds in two districts of the Karamoja Region in north-eastern Uganda. A duplex quantitative real-time polymerase chain reaction (qPCR) assay was used to detect and determine the prevalence of A. marginale (targeting the msp1β gene) and A. centrale (targeting the groEL gene). The qPCR assay revealed that most cattle (82.9%; 95% confidence interval [CI] 78.2–87.7%) were positive for A. marginale DNA, while fewer cattle (12.1%; 95% CI 7.9–16.2%) were positive for A. centrale DNA. A mixed effects logistic regression model showed that the age of cattle was significantly associated with A. centrale infection, while the prevalence of A. marginale varied significantly according to locality. The near full-length 16S ribosomal RNA (16S rRNA) gene and the heat shock protein gene, groEL, for both Anaplasma species were amplified from a selection of samples. The amplicons were cloned and the resulting recombinants sequenced. We found three novel A. marginale 16S rRNA variants, seven A. marginale groEL gene sequence variants and two A. centrale groEL gene sequence variants. Phylogenetic trees were inferred from sequence alignments of the 16S rRNA gene and GroEL amino acid sequences determined here and published sequences using maximum likelihood, Bayesian inference and parsimony methods Phylogenetic analyses classified the 16S rRNA gene and GroEL amino acid sequences into one clade for A. marginale and a separate clade for A. centrale. This study reveals a high prevalence and sequence variability of A. marginale and A. centrale, and is the first report on the phylogenetic characterisation of A. marginale and A. centrale from cattle in Uganda using molecular markers. Sequence variation can be attributed to mobile pastoralism, communal grazing and grazing with wildlife. These data support future epidemiological investigations for bovine anaplasmosis in Uganda.The National Agricultural Research Organisation (NARO), Uganda (P.109224) and University of Pretoria, South Africa (Postgraduate bursary 13399650).http://www.elsevier.com/locate/ttbdis2019-03-01hj2018Veterinary Tropical Disease

Ticks and tick-borne pathogens infecting livestock and dogs in Tchicala-Tcholoanga, Huambo Province, Angola

The diversity of ticks and tick-borne pathogens (TBPs) infesting domestic animals in Tchicala-Tcholoanga, Angola, in 2016 was investigated. Seventeen tick species were recorded, Amblyomma pomposum being the most abundant on cattle (40%), goats (38%) and sheep (35%); Rhipicephalus turanicus was the most abundant on dogs (46%). This study presents new records of Haemaphysalis paraleachi, R. compositus, R. kochi and R. sulcatus in Angola, the first georeferenced population of Ha. leachi in southern Africa and the second record of R. microplus in Angola. Using the reverse line blot (RLB) hybridisation assay, fifteen TBP species were detected in blood samples from cattle (n = 88), goats (n = 82), sheep (n = 85) and dogs (n = 85). F The most frequently detected species were Theileria velifera in cattle (78%), Theileria ovis in sheep (80%) and Babesia vogeli in dogs (35%). Species-specific quantitative PCR assays detected Babesia bigemina in 43% (35/80) of blood samples of cattle, while E. ruminantium was detected in 4% (3/70) of blood samples and in 7% of A. pomposum ticks. Anaplasma platys was detected from cattle (18%) and sheep (6%) during RLB analysis. These findings constitute pioneering research in Angola.The Faculdade de Medicina Veterinária do Huambo, Angola and the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa.http://link.springer.com/journal/436hj2022Veterinary Tropical Disease

Potential hybridization of Fasciola hepatica and F. gigantica in Africa

SUPPLEMENTARY MATERIALS : TABLE S1: Summary of African studies reporting on the presence of Fasciola species recovered from various definitive hosts. TABLE S2: The distribution and occurrence of Fasciola species in Africa based on studies conducted from 1980–2022. TABLE S3: Summary of African studies reporting on the occurrence of Fasciola species in their snail intermediate hosts. TABLE S4: Summary of studies reporting on the occurrence of intermediate hosts of Fasciola spp. in Africa. References [140–317] are cited in the supplementary materials.The occurrence of Fasciola gigantica and F. hepatica in Africa is well documented; however, unlike in Asia, there is a paucity of information on the existence of hybrids or parthenogenetic species on the continent. Nonetheless, these hybrid species may have beneficial characteristics, such as increased host range and pathogenicity. This study provides evidence of the potential existence of Fasciola hybrids in Africa. A literature search of articles published between 1980 and 2022 was conducted in PubMed, Google Scholar, and Science Direct using a combination of search terms and Boolean operators. Fasciola species were documented in 26 African countries with F. hepatica being restricted to 12 countries, whilst F. gigantica occurred in 24 countries, identified based on morphological features of adult Fasciola specimens or eggs and molecular techniques. The cooccurrence of both species was reported in 11 countries. However, the occurrence of potential Fasciola hybrids was only confirmed in Egypt and Chad but is suspected in South Africa and Zimbabwe. These were identified based on liver fluke morphometrics, assessment of the sperms in the seminal vesicle, and molecular techniques. The occurrence of intermediate host snails Galba truncatula and Radix natalensis was reported in Ethiopia, Egypt, South Africa, Tanzania, and Uganda, where F. hepatica and F. gigantica co-occurrences were reported. The invasive Pseudosuccinea columella snails naturally infected with F. gigantica were documented in South Africa and Egypt. In Zimbabwe, P. columella was infected with a presumed parthenogenetic Fasciola. This suggests that the invasive species might also be contributing to the overlapping distributions of the two Fasciola species since it can transmit both species. Notwithstanding the limited studies in Africa, the potential existence of Fasciola hybrids in Africa is real and might mimic scenarios in Asia, where parthenogenetic Fasciola exist in most Asian countries. In South Africa, aspermic F. hepatica and Fasciola sp. have been reported already, and Fasciola hybrids have been reported? in Chad and Egypt. Thus, the authors recommend future surveys using molecular markers recommended to identify Fasciola spp. and their snail intermediate hosts to demarcate areas of overlapping distribution where Fasciola hybrids and/or parthenogenetic Fasciola may occur. Further studies should also be conducted to determine the presence and role of P. columella in the transmission of Fasciola spp. in these geographical overlaps to help prevent parasite spillbacks.The European Union’s Horizon 2020 research and innovation program, the National Research Foundation (NRF) of South Africa, the Knud Højgaards Foundation for its support to The Research Platform for Disease Ecology, Health and Climate.https://www.mdpi.com/journal/pathogensam2023Veterinary Tropical Disease

Characterization of Anaplasma marginale subsp. centrale strains by use of msp1aS genotyping reveals a wildlife reservoir

Bovine anaplasmosis caused by the intraerythrocytic rickettsial pathogen Anaplasma marginale is endemic in South Africa. Anaplasma marginale subspecies centrale also infects cattle; however, it causes a milder form of anaplasmosis and is used as a live vaccine against A. marginale. There has been less interest in the epidemiology of A. marginale subsp. centrale, and, as a result, there are few reports detecting natural infections of this organism. When detected in cattle, it is often assumed that it is due to vaccination, and in most cases, it is reported as coinfection with A. marginale without characterization of the strain. A total of 380 blood samples from wild ruminant species and cattle collected from biobanks, national parks, and other regions of South Africa were used in duplex real-time PCR assays to simultaneously detect A. marginale and A. marginale subsp. centrale. PCR results indicated high occurrence of A. marginale subsp. centrale infections, ranging from 25 to 100% in national parks. Samples positive for A. marginale subsp. centrale were further characterized using the msp1aS gene, a homolog of msp1 of A. mar-ginale, which contains repeats at the 5= ends that are useful for genotyping strains. A total of 47 Msp1aS repeats were identified, which corresponded to 32 A. marginale subsp. centrale genotypes detected in cattle, buffalo, and wildebeest. RepeatAnalyzer was used to examine strain diversity. Our results demonstrate a diversity of A. marginale subsp. centrale strains from cattle and wildlife hosts from South Africa and indicate the utility of msp1aS as a genotypic marker for A. marginale subsp. centrale strain diversity.http://jcm.asm.org2017-04-30hb2017Veterinary Tropical Disease