SSR-based genetic mapping of QTLs determining chilling requirements for time of initial vegetative budbreak in domesticated apple (Malus x domestica Borkh.) cultivar ‘Anna’ x ‘Austin’

>Magister Scientiae - MScThe Rosaceae family contains major temperate crops such as the domesticated apple(Malus x domestica Borkh.), peach (Prunus persica L. Batsch) and European pear (Pyrus communis L.). However, despite its evident economic importance, it is generally poorly studied in genomic terms, relative to the other major crop groups. Microsatellite and Diversity Array Technology (DArT) genetic markers have been exploited in this work and are essential tools in genetic map construction and marker-assisted selection (MAS) of high quality apples and other rosaceous crops. Microsatellites are advantageous in that they are co-dominant, highly polymorphic, abundant, transferable and reliably reproducible; hence their use in this study. In order for budbreak to take place in a timely and homogenous fashion, apple trees need a period of exposure to low temperatures.Within orchards the application of chemicals that induce budbreak in unsuitable environments is required to produce apples from cultivars that require high chilling levels. However, this and other practices using chemicals in orchards tend to pollute the environment. One of the solutions to this problem is to breed low chill apples such as ‘Anna’ cultivar, which was used as one of the parents in this study.This work was aimed at understanding the underlying genetic factors that determine chilling requirements for the time of initial vegetative budbreak trait in the apple cross ‘Anna’ x ‘Austin’. This was achieved through linkage map construction using SSR and DArT molecular markers followed by QTL analysis. This thesis has therefore exploited the large number of Expressed Sequence Tags (ESTs) and genome sequence data for the apple, using Tandem Repeats Finder, to design a total of 98 new SSR primers pairs. The other 369 SSR markers used in this work were from published work. JoinMap! 4.1 software was used to create an integrated genetic map with 17 linkage groups, for the domesticated apple cultivar, ‘Austin’ x ‘Anna’ mapping population with 80 individuals.The result of this process was a genetic map 1 212cM in length, and a total of 429 markers (314 DArT and 115 SSR), at an average density of a marker every 4 cM. This map was used identify the Quantitative Trait Loci (QTLs) determining chilling requirements for time of vegetative budbreak (IVB). In this process, putative IVB QTLs were identified in the ‘Anna’ x ‘Austin’ mapping population using the rMQM analysis function of MapQTL! 6.0, for both adult and seedling data collected over 3 growing seasons from 1996 to 1998. These QTLs were detected on linkage groups 2, 9 and 14,and explained 0.3 to 12.8 % of the observed phenotypic variation for the adult population,and 5.3 - 21 % for the seedling population. Seedling (LG 14) and adult (LGs 5, 7, 10) specific QTLs were also detected for the ‘Anna’ x ‘Austin’ cross. These QTLs will provide the basis for marker validation on related mapping populations in the apple breeding programme, and for the future identification of candidate genes controlling the process of budbreak

Anaplasma marginale outer membrane protein vaccine candidates are conserved in North American and South African strains

Bovine anaplasmosis is a globally economically important tick-borne disease caused by the obligate intraerythrocytic rickettsia, Anaplasma marginale. A live Anaplasma centrale blood-based vaccine is available, but it does not protect against all A. marginale field strains and may also transmit other blood-borne pathogens. Five potential outer membrane protein (OMP) vaccine candidates have been well-characterised in A. marginale strains from the USA, however, their levels of conservation in other countries must be ascertained in order to inform their use in a vaccine with regional or global efficacy. This study assessed the amino acid variation in vaccine candidate OMPs in South African strains of A. marginale, and also compared the immunogenic properties between South African and US strains. OMP genes Am779, Am854, omp7, omp8 and omp9 were amplified and sequenced from a set of genetically diverse South African samples with different msp1α-genotypes. OMPs Am854 and Am779 were highly conserved, with 99–100 % amino acid identity, while Omp7, Omp8 and Omp9 had 79–100 % identity with US strains. As has been shown previously, Omp7–9 possess conserved N- and C- termini, a central variable region, and a highly conserved CD4 T-cell epitope, FLLVDDA(I/V)V, in the N-terminal region. Western blot analysis of recombinant OMPs indicates strong antigenic conservation between South African and US strains of A. marginale, suggesting that they are good candidates for use in a novel global vaccine cocktail, although further work on the best formulation and delivery methods will be necessary.The National Research Foundation (NRF) (Nicola Collins, grant number 81840) and Technology Innovation Agency, Tshwane Animal Health Cluster (Marinda Oosthuizen, grant number TAHC12-00037).http://www.elsevier.com/locate/ttbdis2021-04-18hj2020Veterinary 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

Comparison of three nucleic acid-based tests for detecting Anaplasma marginale and Anaplasma centrale in cattle

Several nucleic acid-based assays have been developed for detecting Anaplasma marginale and Anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas difficult. We evaluated the ability of the reverse line blot (RLB) hybridisation assay, two nested polymerase chain reaction (nPCR) assays and a duplex real-time quantitative polymerase chain reaction (qPCR) assay to detect A. marginale and A. centrale infections in cattle (n = 66) in South Africa. The lowest detection limits for A. marginale plasmid DNA were 2500 copies by the RLB assay, 250 copies by the nPCR and qPCR assays and 2500, 250 and 25 copies of A. centrale plasmid DNA by the RLB, nPCR and qPCR assays respectively. The qPCR assay detected more A. marginale- and A. centrale-positive samples than the other assays, either as single or mixed infections. Although the results of the qPCR and nPCR tests were in agreement for the majority (38) of A. marginale-positive samples, 13 samples tested negative for A. marginale using nPCR but positive using qPCR. To explain this discrepancy, the target sequence region of the nPCR assay was evaluated by cloning and sequencing the msp1β gene from selected field samples. The results indicated sequence variation in the internal forward primer (AM100) area amongst the South African A. marginale msp1β sequences, resulting in false negatives. We propose the use of the duplex qPCR assay in future studies as it is more sensitive and offers the benefits of quantification and multiplex detection of both Anaplasma spp.The National Research Foundation (NRF) of South Africa (grant number 81840 awarded to Dr Nicola Collins) and Technology Innovation Agency (TIA), Tshwane Animal Health Cluster (grant TAHC12-00037 awarded to Professor Marinda Oosthuizen).http://www.ojvr.org/am2017GeneticsVeterinary Tropical Disease

Molecular characterisation of potential vaccine candidates from Anaplasma marginale strains in South Africa

Bovine anaplasmosis, a tick-borne disease caused by the rickettsia Anaplasma marginale, has a significant economic impact for cattle farmers in South Africa. We have estimated the economic cost due to mortality arising from bovine anaplasmosis in the country to be R115 million ($US9.6 million) per year. Further costs are attributable to chemotherapeutic treatment and tick control using acaricides. Anaplasma centrale is a species that is closely related to A. marginale, and in most cases causes a milder, less virulent form of anaplasmosis. It provides cross protection against field strains of A. marginale infection and is therefore employed as a blood vaccine against bovine anaplasmosis in some countries, including South Africa. Despite the economic impact of this disease, there are few studies on the prevalence and control of bovine anaplasmosis in South Africa. This study was therefore carried out in order to evaluate the presence and genetic diversity of A. marginale in the country using quantitative real-time PCR (qPCR) and repeat variation of MSP1a, respectively. In a comprehensive examination of 517 bovine samples from all provinces of South Africa, using the A. marginale and A. centrale qPCR, A. marginale was detected in 57% of our test samples. The rickettsial pathogen was present in all provinces of South Africa with the exception of the Northern Cape province where the tick vector is absent. However, due to recently reported range extension of the important anaplasmosis tick vector Rhiphicephalus microplus, it is believed that this situation may change, and needs close monitoring. Anaplasma centrale was also detected in 17% of the samples, with 15% of the samples being co-infected. An analysis of A. marginale strains present in the samples revealed high genetic diversity, as reflected by the 190 genotypes derived from 99 Msp1a amino acid repeats. This genetic diversity is attributable to a high rate of evolution. Our data also reveal that 22% of the 99 amino acid repeats and, interestingly, only 2 genotypes we found in South Africa, were shared with other countries around the world. Because this study is centred on contributing to the development of a recombinant subunit vaccine, this strain variation should be taken into account in such an undertaking. The current A. centrale blood vaccine has some drawbacks, the two main problems being that it does not protect against heterologous challenge with field strains of A. marginale and it may contribute to transmitting other emerging diseases resulting from a contaminated blood vaccine. Outer membrane protein (OMP) preparations are known to induce immune protection in nearly all animals tested, thus demonstrating the potential efficacy of a subunit vaccine. Five potential OMP vaccine candidates Am779, Am854, Omp7, Omp8 and Omp9 were identified from North American A. marginale strains and have been well-characterised in A. marginale strains from United States of America (USA), but their levels of conservation in other countries were not known. This information would be needed to show that they could be used in a vaccine formulation for a broad application to control bovine anaplasmosis worldwide or in making specific vaccine formulations well-suited for geographic regional strains. In this study, we demonstrated the amino acid variation in these five vaccine candidate OMPs in South African A. marginale strains. We also assessed the immunogenic relationships between South African recombinant versions of these OMPs, and the extensively studied St. Maries and Florida A. marginale strains, from USA. OMPs Am854 and Am779 were found to be highly conserved, with 99–100% amino acid identity. Omp7, Omp8 and Omp9 were also found to be conserved with 79–100% identity with St. Maries and Florida strains. We also found, as has been shown previously, that the latter OMPs possess conserved N- and Cterminals, along with a pronounced, central hypervariable region. A previously identified, highly conserved T-cell epitope, FLLVDDAI/V, was also found in the conserved Nterminus of these three OMPs. Through recognition of South African recombinant OMPs by anti-A. marginale and A. centrale bovine sera from South Africa and USA, we were able to demonstrate immunological cross-reactivity between the A. centrale and A. marginale organisms. This suggests that there are significant antigenic and immunological relationships between South Africa and USA strains of A. marginale, and provides evidence for the continued use of the A. centrale blood vaccine for immunisation against A. marginale infections. Our study also provides evidence to suggest that the A. marginale OMPs are good vaccine candidates for use in a global vaccine cocktail, although further work on the best formulation and delivery methods is necessary. For the purposes of creating a biobank of A. marginale strains for downstream ‘-omic’ studies and to provide challenge material for future vaccine trials, we attempted to culture field strains of A. marginale from 17 bovine blood samples in ISE6 and IDE8 tick cell lines. Blood from three persistently infected and 12 clinically sick animals was used to attempt direct infection of tick cell lines, but yielded negative cultures after approximately 160 days in culture. We therefore attempted to initiate cultures using blood from two splenectomised calves that were infected with blood from A. marginale-carrier animals. These have yielded promising results as small colonies could be observed after about 60 days of culture, and DNA extracts of these cultures were qPCR-positive. We will continue to monitor these cultures by Giemsa staining, light microscopy and qPCR, for progression of the infection.Veterinary Tropical DiseasesPhDUnrestricte

Detection and Characterisation of Anaplasma marginale and A. centrale in South Africa

Bovine anaplasmosis is endemic in South Africa and it has a negative economic impact on cattle farming. An improved understanding of Anaplasma marginale and Anaplasma marginale variety centrale (A. centrale) transmission, together with improved tools for pathogen detection and characterisation, are required to inform best management practices. Direct detection methods currently in use for A. marginale and A. centrale in South Africa are light microscopic examination of tissue and organ smears, conventional, nested, and quantitative real-time polymerase chain reaction (qPCR) assays, and a reverse line blot hybridisation assay. Of these, qPCR is the most sensitive for detection of A. marginale and A. centrale in South Africa. Serological assays also feature in routine diagnostics, but cross-reactions prevent accurate species identification. Recently, genetic characterisation has confirmed that A. marginale and A. centrale are separate species. Diversity studies targeting Msp1a repeats for A. marginale and Msp1aS repeats for A. centrale have revealed high genetic variation and point to correspondingly high levels of variation in A. marginale outer membrane proteins (OMPs), which have been shown to be potential vaccine candidates in North American studies. Information on these OMPs is lacking for South African A. marginale strains and should be considered in future recombinant vaccine development studies, ultimately informing the development of regional or global vaccines

Co-infections with multiple genotypes of Anaplasma marginale in cattle indicate pathogen diversity

BACKGROUND : Only a few studies have examined the presence of Anaplasma marginale and Anaplasma centrale in South Africa, and no studies have comprehensively examined these species across the whole country. To undertake this country-wide study we adapted a duplex quantitative real-time PCR (qPCR) assay for use in South Africa but found that one of the genes on which the assay was based was variable. Therefore, we sequenced a variety of field samples and tested the assay on the variants detected. We used the assay to screen 517 cattle samples sourced from all nine provinces of South Africa, and subsequently examined A. marginale positive samples for msp1α genotype to gauge strain diversity. RESULTS : Although the A. marginale msp1β gene is variable, the qPCR functions at an acceptable efficiency. The A. centrale groEL gene was not variable within the qPCR assay region. Of the cattle samples screened using the assay, 57% and 17% were found to be positive for A. marginale and A. centrale, respectively. Approximately 15% of the cattle were co-infected. Msp1α genotyping revealed 36 novel repeat sequences. Together with data from previous studies, we analysed the Msp1a repeats from South Africa where a total of 99 repeats have been described that can be attributed to 190 msp1α genotypes. While 22% of these repeats are also found in other countries, only two South African genotypes are also found in other countries; otherwise, the genotypes are unique to South Africa. CONCLUSIONS : Anaplasma marginale was prevalent in the Western Cape, KwaZulu-Natal and Mpumalanga and absent in the Northern Cape. Anaplasma centrale was prevalent in the Western Cape and KwaZulu-Natal and absent in the Northern Cape and Eastern Cape. None of the cattle in the study were known to be vaccinated with A. centrale, so finding positive cattle indicates that this organism appears to be naturally circulating in cattle. A diverse population of A. marginale strains are found in South Africa, with some msp1α genotypes widely distributed across the country, and others appearing only once in one province. This diversity should be taken into account in future vaccine development studies.The National Research Foundation (NRF) (Nicola Collins, grant number 81840); Technology Innovation Agency, Tshwane Animal Health Cluster (TIA, TAHC) (Marinda Oosthuizen, grant number TAHC12-00037).http://www.parasitesandvectors.comam2018Forestry and Agricultural Biotechnology Institute (FABI)Veterinary Tropical Disease

Comparison of three nucleic acid-based tests for detecting <i>Anaplasma marginale</i> and <i>Anaplasma centrale</i> in cattle

Several nucleic acid-based assays have been developed for detecting Anaplasma marginale and Anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas difficult. We evaluated the ability of the reverse line blot (RLB) hybridisation assay, two nested polymerase chain reaction (nPCR) assays and a duplex real-time quantitative polymerase chain reaction (qPCR) assay to detect A. marginale and A. centrale infections in cattle (n = 66) in South Africa. The lowest detection limits for A. marginale plasmid DNA were 2500 copies by the RLB assay, 250 copies by the nPCR and qPCR assays and 2500, 250 and 25 copies of A. centrale plasmid DNA by the RLB, nPCR and qPCR assays respectively. The qPCR assay detected more A. marginale- and A. centrale-positive samples than the other assays, either as single or mixed infections. Although the results of the qPCR and nPCR tests were in agreement for the majority (38) of A. marginale-positive samples, 13 samples tested negative for A. marginale using nPCR but positive using qPCR. To explain this discrepancy, the target sequence region of the nPCR assay was evaluated by cloning and sequencing the msp1β gene from selected field samples. The results indicated sequence variation in the internal forward primer (AM100) area amongst the South African A. marginale msp1β sequences, resulting in false negatives. We propose the use of the duplex qPCR assay in future studies as it is more sensitive and offers the benefits of quantification and multiplex detection of both Anaplasma spp