Project topics and materials
Get Complete Project Material File(s) Now! »
Characterization of Anaplasma marginale subspecies centrale strains using Msp1aS genotyping reveals a wildlife reservoir
Abstract
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 co-infection with A. marginale without characterization of the strain. In this study 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 25-100% in National Parks. Samples positive for A. marginale subsp. centrale were further characterized using the msp1aS gene, a homolog of msp1α of A. marginale which contains repeats at the 5’end 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.
Introduction
Bovine anaplasmosis (gallsickness) is a tick-borne disease caused by the intra-erythrocytic rickettsial pathogen Anaplasma marginale (Theiler, 1910). Anaplasma marginale is globally prevalent and results in anemia, with mortality rates of up to 30% (Losos, 1986). Anaplasma marginale subspecies centrale, is a less virulent subspecies detected by Sir Arnold Theiler, who recognized its potential as a vaccine against anaplasmosis; 100 years later this live vaccine is still in use in South Africa, Israel, South America and Australia (Theiler, 1911; Aubry & Geale, 2011). The strain that is used as a vaccine originated from Theiler’s original isolation and was exported at various times to other countries where it has been propagated in the laboratory; the strain known as the “Israel strain” or the “vaccine strain” was sent to Israel in the 1950s, and was used to generate the complete genome sequence for A. marginale subsp. centrale in 2010 (Herndon et al., 2010). Anaplasma marginale subsp. centrale does not provide complete protection against A. marginale infection, but does protect against severe anaplasmosis (Kuttler, 1984; Anziani et al., 1987).
Anaplasma marginale infects a wide range of ruminants including buffalo (Bubalus bubalis and Syncerus caffer), wildebeest (Connochaetes gnou and Connochaetes taurinus), American bison (Bison bison), white-tailed deer (Odocoileus virginianus), mule deer (Odocoileus hemionus hemionus), black-tailed deer (Odocoileus hemionus columbianus), and Rocky Mountain elk (Cervus elaphus nelsoni) (Neitz, 1935; Potgieter, 1979; Smith et al., 1982; Potgieter & Stoltsz, 2004 ). Cattle are naturally susceptible to A. marginale (Aubry & Geale, 2011). There has not been much interest in the epidemiology of A. marginale subsp. centrale, with few reports detecting natural infections of this organism; most often, when detected in cattle it is assumed that it is due to vaccination and is reported as co-infection with A. marginale without characterization of the strain. Georges et al. (2001) reported A. marginale subsp. centrale single infections detected by the reverse line blot (RLB) hybridization assay in Italy without characterizing the strain. More recently, the first known case of bovine anaplasmosis caused by A. marginale subsp. centrale in Europe was reported (Carelli et al., 2008). While this study described genetic heterogeneity of A. marginale subsp. centrale strains from different geographic areas in Italy, it is not clear how these are related to the vaccine strain.
For A. marginale, the Msp1a protein/gene (msp1α) has been used as a genotypic marker to differentiate strains (Allred et al., 1990). Msp1a is encoded by the single copy gene, msp1α, and differs among strains due to variable sequence and numbers of a 28 or 29 amino acid (84/87-bp) sequence repeat located near the amino-terminus of the protein (Allred et al., 1990). A number of studies have examined Msp1a repeats in the USA, South America, Australia, the Philippines, Europe, Israel, China and Mexico resulting in identification of over 200 repeats (Allred et al., 1990; Bowie et al., 2002; de la Fuente et al., 2007). In South Africa, two studies have been conducted to genetically characterize strains using msp1α (Mutshembele et al., 2014; Mtshali et al., 2007), revealing that the repeat structure is common between South African, American and European strains of A. marginale; in fact, some of the repeat sequences that were detected were identical to ones that were detected in the USA. Not surprisingly, there were also new repeat sequences detected that are, thus far, unique to South Africa.
A marginale subsp. centrale was thought not to have a homolog of msp1α, however, complete genome sequencing of the Israel vaccine strain revealed that there is a gene that resides in a syntenic position to A. marginale msp1α (Herndon et al., 2010). This gene was named msp1aS (S for syntenic; a gene flanked by the same set of genes in two genomes), and has 31-36% amino acid sequence identity depending on the A. marginale strain compared. Importantly, there are structural similarities, including repeats near the amino terminus and two sets of transmembrane domains near the carboxy-terminus that indicate that these proteins are likely homologs (Fig. 3.1). The repeats in A. marginale subsp. centrale strain Israel Msp1aS are longer (47 amino acids in length) than the A. marginale Msp1a repeats and there is no sequence identity between the repeats in the two organisms. The vaccine strain (abbreviated as “Ac”) has four repeats with an msp1aS genotype of Ac1 Ac1 Ac1 Ac2 (with the number indicating the tandem repeat type).
In the present study, we have used a duplex qPCR assay to screen for the presence of A. marginale subsp. centrale and A. marginale in vaccinated and unvaccinated cattle and wildlife indicating that these infections are common and often occur as mixed infections. Samples that tested positive using this screen were then further analyzed for msp1aS genotype, demonstrating that the vaccine strain genotype is prevalent in cattle herds that practice vaccination while other more divergent genotypes are present in wildlife species.
Materials and Methods
Blood collection and DNA extraction
A total of 380 blood samples from wild ruminant species including African buffalo (n=97) (Syncerus caffer), waterbuck (n=14) (Kobus ellipsipyrymnus), eland (n=23) (Taurotragus oryx), black wildebeest (n=54) (Connochaetes gnou) and blue wildebeest (n=23) (Connochaetes taurinus) together with 86 cattle samples were obtained from the Wildlife Biological Resource Center (WBRC) and Biobank South Africa under the auspices of the National Zoological Gardens of South Africa (NZG) as well as from the South African National Parks (SANParks) Biobank. The remaining buffalo blood samples (n=41), were made available to us by Dr. Dave Cooper from Hluhluwe-iMfolozi Park. Additionally, 42 blood samples from vaccinated cattle were obtained from two commercial farms in Bergville, KwaZulu-Natal, South Africa (Table 3.1). Standard techniques were followed in collecting blood samples for laboratory examination. Genomic DNA was extracted using the QIAmp DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA was eluted in 100 μl elution buffer and stored at -20°C. The study was approved by the Animal Ethics Committee of the University of Pretoria, South Africa (V085-14) and permission to use wildlife samples was given by SANParks Biobank under reference number “LARBJ1118 Conservation Genetics”, the WBRC, and Biobank SA under the auspices of the NZG of South Africa and the Johannesburg Zoo with project number NZG/P13/05. Collection of cattle samples was approved by the Department of Agriculture Forestry and Fisheries under section 20 of the Animal Diseases Act of 1984 with reference 12/11/1/1.
Duplex real-time PCR assay
Quantitative real-time PCR (qPCR) for simultaneous detection and quantification of A. marginale and A. marginale subsp. centrale DNA was performed as described previously (Decaro et al., 2008) with some modifications for use on a Light Cycler real-time machine (Chaisi et al., 2017) (Roche Diagnostics, Mannheim, Germany). The qPCR was performed in a final reaction volume of 20 μl, containing 2 μl of DNA template (100-200 ng of DNA), 12.5 μl of FastStart DNA Master Hybridization kit (Roche Diagnostics, Mannheim, Germany), 600 nM of A. marginale-specific primers AM-For (5’ TTG GCA AGG CAG CAG CTT 3’) and AM-Rev (5’ TTC CGC GAG CAT GTG CAT 3’), 900 nM of A. marginale subsp. centralespecific primers AC-For (5’ CTA TAC ACG CTT GCA TCT C 3’) and AC-Rev (5’ CGC TTT ATG ATG TTG ATG C 3’) and 200 nM of probes AM-Pb (5’ 6FAM-TCG GTC TTA ACA TCT CCA GGC TTT CAT-BHQ1 3’) and AC-Pb (5’ LC610-ATC ATC ATT CTT CCC CTT TAC CTC GT-BHQ2 3’). Thermal cycling conditions were: UDG activation at 40°C for 10 min, pre-incubation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 1 min and annealing-extension at 60°C for 1 min, and a final cooling step at 40°C for 30 sec. The results were analyzed using the Lightcycler Software version 4.0 (Roche Diagnostics, Mannheim, Germany). The software indicates a positive result by a Cq value (quantification cycle, synonymous with the Cp, crossing point, value given by the Lightcycler instrument), at which fluorescence from amplification exceeds the background fluorescence, and a score of 1 to 5. Negative samples have a score of -1 to -5 and no Cp values. A lower Cq correlates with a higher starting concentration of target DNA in a sample, which then indicates a positive infection. FAM fluorescence (530 nm) was generated in A. marginale positive samples and LC-610 (610 nm) signals were generated in A. marginale subsp. centrale positive samples. Deoxyribonucleic acid extracted from the A. marginale subsp. centrale vaccine strain (Onderstepoort Biological Products, Pretoria) was used as a positive control, and samples C14, C57 or F48 (originating from cattle in the Mnisi Community area, Mpumalanga, South Africa) were used as positive controls for A. marginale. The presence of A. marginale in these samples was confirmed by sequencing of the msp1β genes. A negative and positive control was included in each set of PCR reactions that was performed. The analytical specificity of the assay was determined by analyzing DNA from closely related species such as Anaplasma sp. Omatjenne and A. phagocytophilum (Carelli et al., 2007). The efficiency of the assay was determined from 10-fold serial dilutions of plasmid DNA from clones 9410c (A. marginale subsp. centrale) and F48a (A. marginale).
Analysis of the msp1aS gene
Anaplasma marginale subsp. centrale-positive samples which had low Cq values as detected by qPCR were selected for analysis of the msp1aS gene. Primers MSP1asFZ (5’ CAA GGT CAA GAG TCA GCA TCA TCA GAT G 3’) and MSP1asRZ (5’ CTC CGC GCA CAA TAC TTT CAA CCT CC 3’) were designed based on the A. marginale subsp. centrale genome sequence (Genbank accession # CP001759) to target tandem repeats within the msp1aS gene. PCR was performed in a final reaction volume of 25 µl containing Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific), 10 pM of each primer and genomic DNA. Thermal cycling was carried out in a Veriti thermal cycler (Thermo Fisher Scientific), and consisted of an initial denaturation at 98°C for 10 sec, followed by 30 cycles of denaturation at 98°C for 1 sec, annealing at 67°C for 30 sec and extension at 72°C for 15 sec, and a final extension at 72°C for 1 min. DNA extracted from the A. marginale subsp. centrale vaccine obtained from Onderstepoort Biological Products (OBP, Pretoria, South Africa) was used as a positive control.
Purified PCR amplicons were cloned into the pJET vector (Thermo Fisher Scientific). Recombinant plasmids were isolated using a High Pure Plasmid Isolation Kit (Roche Diagnostics, Mannheim, Germany) and sequenced using 1 µl of 2 µM M13 primers with ABI Big Dye V3.1 Kit on an ABI 3500XL genetic analyzer at Inqaba Biotec (Pretoria, South Africa).
Sequences were assembled, edited, and translated to amino acids using CLC Main Workbench 7.0.3 (Qiagen, Denmark). Tandem repeats were identified using Tandem Repeats Finder (https://tandem.bu.edu/trf/trf.html) (Benson, 1999). The repeats were named Ac#, to distinguish them from A. marginale Msp1a repeats. Truncated repeats were designated with a T at the end of the name. Repeats were curated and analyzed using RepeatAnalyzer (Catanese et al., 2016). Repeat sequences were aligned using the AlignX module of Vector NTI (Invitrogen).
Diversity Measures
RepeatAnalyzer calculates four genetic diversity metrics, each of which captures the diversity of repeats in a geographic region in a different way. Broadly, they fall into two groups, those that measure the amount of different repeats and those that measure the distribution of those repeats. Within each of these categories, there is a global and a local formulation. The local version of a metric calculates the score independently on each genotype and averages these together to get the final score, while the global version looks at all genotypes together. Specifically, the GDM1L score can be interpreted as the percent of unique repeats in each genotype in the region, while the GDM1G score is the percent of unique repeats across all genotypes in the region. The GDM2L score can be interpreted as the amount of variation (measured as standard deviation) in the number of occurrences of the repeats in a genotype, while the GDM2G score is the amount of variation in the number of occurrences of all the repeats in all genotypes in the region. A high GDM1 score means that there are more unique repeats, with 0 as the minimum (when all repeats are the same) and 1 being the maximum (when each repeat is unique). A high GDM2 score means that the repeats are distributed more unevenly, with a minimum of 0 (when all repeats occur the same number of times) and values ranging up to but not including 0.5 as the unevenness of repeat distribution increases.
DECLARATION
ACKNOWLEDGEMENTS
DEDICATION
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF TABLES
LIST OF ABBREVIATIONS
THESIS SUMMARY
CHAPTER 1 General Introduction
1.1 Background
1.2 Problem Statement
1.3 Objectives
1.4 Thesis overview
1.5 References
CHAPTER 2 Literature Review
2.1 History and taxonomic position
2.2 Transmission and arthropod vectors
2.3 Epidemiolog
2.3.1 Geographical distribution
2.3.2 Reservoir hosts
2.4 Clinical signs and diagnosis
2.5 Treatment, prevention and control strategies
2.6 Anaplasma major surface proteins and their role in host-vector-pathogen interactions
2.7 References
CHAPTER 3 Characterization of Anaplasma marginale subspecies centrale strains using Msp1aS genotyping reveals a wildlife reservoir
3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.3.1 Blood collection and DNA extraction
3.3.2 Duplex real-time PCR assay
3.3.3 Analysis of the msp1aS gene
3.3.4 Diversity Measures
3.4 Results
3.4.1 Occurrence of Anaplasma species in wild ruminants and cattle in South Africa
3.4.2 Characterization of Msp1aS
3.4.3 Diversity analysis and repeat distribution
3.5 Discussion
3.6 Conclusion
3.7 References
CHAPTER 4 Evidence confirming the phylogenetic position of Anaplasma centrale (ex Theiler1911) Ristic & Kreier 1984
4.1 Abstract
4.2 Introduction
4.3 Materials and Methods
4.3.1 Selection of samples for amplification, cloning and sequencing
4.3.2 Amplification of the 16S rRNA, groEL and msp4 genes
4.3.3 Sequencing and phylogenetic analysis of 16S rRNA, groEL and msp4 genes
4.4 Results
4.4.1 16S rRNA, groEL and msp4 gene sequence and phylogenetic analysis
4.5 Discussion
4.5.1 Description of Anaplasma centrale Anaplasma centrale (ex Theiler, 1911) sp. nov., comb. nov. (Ristic & Kreier, 1984)
4.6 References
CHAPTER 5 Anaplasma centrale Msp1aS genotyping: Can it shed light on the possible tick vector(s) of centrale that circulate in the cattle populations in uThukela district, South Africa?
5.1 Abstract
5.2 Introduction
5.3 Materials and Methods
5.3.1 Ethical approval
5.3.2 Study area
5.3.3Collection of ticks and blood samples from cattle
5.3.4 DNA extraction
5.3.5 Duplex qPCR for simultaneous detection of marginale and A. centrale
5.3.6 PCR amplification of the msp1aS gene
5.3.7 Cloning and sequencing of PCR products
5.3.8 Sequence analysis
5.4 Results
5.4.1 Identification of tick species
5.4.2 Duplex qPCR for simultaneous detection of marginale and A. centrale in tick tissues and cattle DNA
5.4.3 PCR amplification of msp1aS gene
5.4.4 Anaplasma centrale msp1aS tandem repeats
5.5 Discussion.
5.6 Conclusion
5.7 References
CHAPTER 6 General Discussion and Conclusions
6.1 References
APPENDICES
Appendices 1-5: Supplementary Tables for Chapter 4
Appendices 6: Supplementary photos for Chapter 5
Appendices 7-9: Ethical Documents
CONFERENCES
MANUSCRIPTS
