Molecular genotyping of Babesia caballi.

Babesia caballi is an intra-erythrocytic parasite causing equine piroplasmosis. Three B. caballi genotypes (A, B, and C) have been identified based on the 18 S rRNA and rhoptry-associated protein (rap-1) gene sequences. These variant parasite genotypes compromise the diagnostic utility of the WOAH-recommended serological assays in declaring horses free of equine piroplasmosis. Although a gene encoding a spherical body protein 4 (sbp4) has recently been identified as a potential antigen for the serological detection of B. caballi, the ability of this antigen to detect the different geographical strains has not been determined. The molecular distinction between variant B. caballi genotypes is limited and therefore we developed molecular typing assays for the rapid detection and quantification of distinct parasite genotypes. Field samples were screened for the presence of B. caballi using an established multiplex equine piroplasmosis qPCR assay. In this study, B. caballi genotype A was not detected in any field samples screened. However, phylogenetic analysis of the amplified sbp4 and 18 S rRNA genes confirmed the phylogenetic groupings of the South African isolates into either B. caballi genotypes B or C. A multiple sequence alignment of the sbp4 gene sequences obtained in this study together with the published sbp4 sequences representing B. caballi genotype A, were used to identify conserved regions within the gene to design three primer pairs and three genotype-specific TaqMan minor-groove binder (MGB™) probes. The qPCR assays were shown to be specific and efficient in the detection and differentiation between B. caballi genotypes A, B, and C and could be used as a diagnostic assay to prevent the unintentional spread of variant B. caballi genotypes globally.

Unraveling the Complexity of the Rhomboid Serine Protease 4 Family of Babesia bovis Using Bioinformatics and Experimental Studies.

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host−pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.

South African Buffalo-Derived Theileria parva Is Distinct From Other Buffalo and Cattle-Derived T. parva.

Theileria parva is a protozoan parasite transmitted by the brown-eared ticks, Rhipicephalus appendiculatus and Rhipicephalus zambeziensis. Buffaloes are the parasite's ancestral host, with cattle being the most recent host. The parasite has two transmission modes namely, cattle-cattle and buffalo-cattle transmission. Cattle-cattle T. parva transmission causes East Coast fever (ECF) and January disease syndromes. Buffalo to cattle transmission causes Corridor disease. Knowledge on the genetic diversity of South African T. parva populations will assist in determining its origin, evolution and identify any cattle-cattle transmitted strains. To achieve this, genomic DNA of blood and in vitro culture material infected with South African isolates (8160, 8301, 8200, 9620, 9656, 9679, Johnston, KNP2, HL3, KNP102, 9574, and 9581) were extracted and paired-end whole genome sequencing using Illumina HiSeq 2500 was performed. East and southern African sample data (Chitongo Z2, Katete B2, Kiambu Z464/C12, Mandali Z22H10, Entebbe, Nyakizu, Katumba, Buffalo LAWR, and Buffalo Z5E5) was also added for comparative purposes. Data was analyzed using BWA and SAMtools variant calling with the T. parva Muguga genome sequence used as a reference. Buffalo-derived strains had higher genetic diversity, with twice the number of variants compared to cattle-derived strains, confirming that buffaloes are ancestral reservoir hosts of T. parva. Host specific SNPs, however, could not be identified among the selected 74 gene sequences. Phylogenetically, strains tended to cluster by host with South African buffalo-derived strains clustering with buffalo-derived strains. Among the buffalo-derived strains, South African strains were genetically divergent from other buffalo-derived strains indicating possible geographic sub-structuring. Geographic sub- structuring was also observed within South Africa strains. The knowledge generated from this study indicates that to date, ECF is not circulating in buffalo from South Africa. It also shows that T. parva has historically been present in buffalo from South Africa before the introduction of ECF and was not introduced into buffalo during the ECF epidemic.

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.

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.

Whole genome sequencing of Theileria parva using target capture.

Protozoan parasite isolation and purification are laborious and time-consuming processes required for high quality genomic DNA used in whole genome sequencing. The objective of this study was to capture whole Theileria parva genomes directly from cell cultures and blood samples using RNA baits. Cell culture material was bait captured or sequenced directly, while blood samples were all captured. Baits had variable success in capturing T. parva genomes from blood samples but were successful in cell cultures. Genome mapping uncovered extensive host contamination in blood samples compared to cell cultures. Captured cell cultures had over 81 fold coverage for the reference genome compared to 0-33 fold for blood samples. Results indicate that baits are specific to T. parva, are a good alternative to conventional methods and thus ideal for genomic studies. This study also reports the first whole genome sequencing of South African T. parva.

Microsatellite and minisatellite genotyping of Theileria parva population from southern Africa reveals possible discriminatory allele profiles with parasites from eastern Africa.

The control of Theileria parva, a protozoan parasite that threatens almost 50% of the cattle population in Africa, is still a challenge in many affected countries. Theileria parva field parasites from eastern Africa, and parasites comprising the current live T. parva vaccine widely deployed in the same region have been reported to be genotypically diverse. However, similar reports on T. parva parasites from southern Africa are limited, especially in Corridor disease designated areas. Establishing the extent of genetic exchange in T. parva populations is necessary for effective control of the parasite infection. Twelve polymorphic microsatellite and minisatellite loci were targeted for genotypic and population genetics analysis of T. parva parasites from South Africa, Mozambique, Kenya and Uganda using genomic DNA prepared from cattle and buffalo blood samples. The results revealed genotypic similarities among parasites from the two regions of Africa, with possible distinguishing allelic profiles on three loci (MS8, MS19 and MS33) for parasites associated with Corridor disease in South Africa, and East Coast fever in eastern Africa. Individual populations were in linkage equilibrium (VDL) was observed. Genetic divergence was observed to be more within (AMOVA = 74%) than between (AMOVA = 26%) populations. Principal coordinate analysis showed clustering that separated buffalo-derived from cattle-derived T. parva parasites, although parasites from cattle showed a close genetic relationship. The results also demonstrated geographic sub-structuring of T. parva parasites based on the disease syndromes caused in cattle in the two regions of Africa. These findings provide additional information on the genotypic diversity of T. parva parasites from South Africa, and reveal possible differences based on three loci (MS8, MS19 and MS33) and similarities between buffalo-derived T. parva parasites from southern and eastern 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 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.

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 ß-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 ß-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.

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.

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.

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.