MOLECULAR DETECTION, GENETIC DIVERSITY AND PHYLOGENETIC ANALYSIS 
OF ANAPLASMA MARG/NALE INFECTING CATTLE IN SOUTH AFRICA 
By 
Awelani Mirinda Mutshembele 
Thesis submitted in fulfillment of the requirements for the degree Philosophiae 
Doctor in the Faculty of Natural and Agricultural Sciences, Department of Zoology 
and Entomology, University of the Free State 
Promoters: Dr. M.S Mtshali and Prof O.M.M Thekisoe 
December 2015 
PROMOTERS 
Dr. Moses S. Mtshali (PhD) 
Research and Scientific Services Department 
National Zoological Gardens of South Africa 
P.O. Box 754 
Pretoria 
0001 
Department of Zoology and Entomology, 
University of the Free State, QwaQwa Campus 
Private Bag X 13 
Phuthaditjhaba 
9866 
Prof. Oriel M.M. Thekisoe (PhD) 
Unit for Environmental Sciences and Management 
North - West University, Potchefstroom Campus 
Private Bag x 6001 
Potchefstroom 
2520 
ii 
DECLARATION 
I, the undersigned, hereby declare that the work contained in this thesis is my own 
original work and that I have not previously in it's entirely or in part submitted it at any 
university for a degree. I furthermore, cede copyright of the thesis in favour of the 
University of the Free State 
.. . . .. ... ~~· ··· · 0.9..l 0.~ . \ .?-9.! .~ ... 
AM. Mutshembele Date 
iii 
DEDICATION 
This thesis is dedicated to my parents my late father Itani Daniel Mutshembele may his 
soul rest in peace, and my mother Tshilidzi Mercy Mutshembele for their endless love, 
support , encouragement and tolerance on my career choice. 
iv 
ACKNOWLEDGEMENTS 
I thank Almighty God for the strength He gave me to do research under supervision of 
Dr. M. S. Mtshali and Prof 0 . M. M. Thekisoe, without their assistance and dedicated 
involvement in every step throughout the process, this study would have never been 
accomplished. I thank them very much for their support and understanding over these 
past four years. I also thank our collaborators Prof Jose de la Fuente and Dr. Alejandro 
Cabezas-Cruz for their endless support during my research period. 
I would also like to show gratitude to my colleagues and friends in the Veterinary 
Parasitology Programme for their immeasurable contributions and not forgett ing the 
soul mates Dr. Raksha Bhoora, Dr. Tracy Masebe, Zama Khumalo, Lesego Modibedi -
Masango, Portia Motheo, Sabath Rathanya, Verdi Giqwa and Seipati Nku for their 
support and patience during difficult study times and "Sissy Khanyi" Prof Khanyisile 
Mbatha for her support and advice throughout the year, she was my pillar. 
I also thank Moeti Oriel Taioe for his assistance with phylogenetic analys is at the 
Department of Zoology and Entomology, University of Free State QwaQwa Campus 
and Dr. Steve Olorunju at the Biostatistics Unit, Medical Research Council of South 
Africa, Pretoria who kindly assisted me with the statistical analysis and was very patient 
with me. 
v 
I thank my brothers Dr. Theodore Mutshembele, Ndivhoni Mutshembele, my sister 
Mudzunga Mutshembele and my nieces (Mapitso and Malindi). Every time I was ready 
to quit, they did not let me to quit and I am forever grateful, this thesis stands as a 
testament to your unconditional love and encouragement, and Abel Ralinala for his love, 
patience, jokes and words of inspiration that gave me strength and kept me motivated. 
Getting through my dissertation required more than academic support, and I have 
many, many people to thank for listening to me and, at times, having to tolerate me over 
the past three years. I cannot begin to express my gratitude and appreciation for their 
friendsh ip. 
I also thank the Directors of Department of Agriculture and Veterinary Institute from 
Limpopo, Mpumalanga, North West, KwaZulu-Natal , Eastern Cape, Western Cape and 
Northern Cape (Prof. Nomfundo Mnisi, Ors Mlilo, Songelwayo Chisi, L. Mrwebi, Ronald 
Sinclair and Francis Sabi) with the assistance from the Veterinary Technicians (Toypat 
Mdlul i, Keneilwe Constance Kaotane, Jan Masethe Maime, Anil Suresh and Erwin 
Lucas) and the farmers who co-operated in sample collection . 
I acknowledge the University of the Free State and National Zoological Gardens of 
South Africa for availing their facilities during this study. This study is based on the 
research supported in part by the National Research Foundation of South Africa from 
the DST/NRF Professional Development Programme, grant made available to Dr. M.S. 
Mtshali; and the National Zoological Gardens of South Africa. Any opinion, find ing and 
conclusion or recommendation expressed in th is study is that of the author and the NRF 
does not accept any liabi lity in th is regard. 
vi 
TABLE OF CONTENTS 
CONTENTS PAGE 
TITLE 
PROMOTERS ii 
DECLARATION iii 
DEDICATION iv 
ACKNOWLEDGEMENTS v 
TABLE OF CONTENTS vii 
LIST OF FIGURES xi 
LIST OF TABLES xv 
APPENDICES xvi 
LIST OF ABBREVIATIONS xvii 
RESEARCH OUTPUTS xix 
ABSTRACT xxi 
1 INTRODUCTION AND LITERATURE REVIEW 
1.1 Historical background 1 
1.2 Classification of Anaplasma marginale 1 
1.3 Epidemiology 2 
1.4 Distribution of ticks infesting cattle in South Africa 4 
1.5 Pathogenesis of A. marginale 7 
1.6 T ra nsm ission 9 
1.7 Life cycle of A. marginale 10 
1.8 Geographic distribution and economic importance of anaplasmosis 12 
1.9 Diagnosis of anaplasmosis 14 
1.9.1 Competetive enzyme -linked immunosorbent assay 16 
1.9.2 Card agglutination test 16 
vii 
1.9.3 Complement fixation test 17 
1.9.4 Indirect fluorescent antibody test 17 
1.9.5 Polymerase chain reaction 17 
1.10 Treatment, prevention and control strategies 19 
1.11 Anaplasma marginale major surface proteins and their role in host-vector- 21 
pathogen interactions 
1.11 .1 The MSP1 complex 22 
1.11 .2 Vector-pathogen relationships 26 
1.11 .2.1 Anaplasma MSPs and vector-pathogen interactions 26 
1.12 Phylogenetic relationships of geographic isolates of A. marginale 27 
2 OBJECTIVES OF THE STUDY 
2.1 Statement of the problem 29 
2.2 Aim of the study 32 
2.3 Objectives 32 
3 MOLECULAR DETECTION OF ANAPLASMA MARG/NALE ISOLATES 
INFECTING CATTLE IN SOUTH AFRICA BY PCR TARGETING msp1a 
GENE 
3.1 Introduction 33 
3.2 Materials and methods 34 
3.2.1 Study site and sample collection 34 
3.2.2 DNA extraction 35 
3.2.3 Polymerase chain reaction (PCR) 35 
3.2.4 Statistical analysis 36 
3.3 Results 37 
3.3.1 Detection of A. margina/e infections 37 
3.3.2 Molecular prevalence of A. margina/e isolates infecting cattle in South 37 
viii 
African provinces 
3.3.3 Statistical prevalence of A. marginale isolates infecting cattle in South 37 
African provinces 
3.4 Discussion 44 
4. EPIDEMIOLOGY AND EVOLUTION OF THE GENETIC VARIABILITY OF 
ANAPLASMA MARG/NALE IN SOUTH AFRICA 
4.1 Introduction 43 
4.2 Materials and methods 45 
4.2.1 Study site and sample collection 45 
4.2.2 DNA extraction 49 
4.2.3 A. marginale species-specific PCR 49 
4.2.4 DNA sequencing of A. marginale msp 1a  gene 49 
4.2.5 Sequence analysis of A. marginale msp1a gene 50 
4.2.6 Codon based phylogenetic analysis of tandem repeats 50 
4.2.7 Amino acid variability, composition and genetic diversity index (GDI) 50 
4.3 Results and discussion 52 
4.3.1 Molecular evidence of A. marginale prevalence in South Africa 52 
4.3.2 A. marginale prevalence and msp1 a genetic diversity 55 
4.3.3 Evolution of msp1a genetic diversity 56 
4.3.4 Amino acid variability and low variable msp1a peptides 64 
5. STRUCTURAL AND PHYLOGENETIC ANALYSIS OF ANAPLASMA 
MARG/NALE INFECTING CATTLE IN SOUTH AFRICA USING msp1a 
AND msp4 GENES 
5.1 Introduction 66 
5.2 Materials and methods 67 
5.2.1 DNA extraction 67 
5.2.2 Set of primers used for amplification of msp 1a  and msp4 genes 68 
5.2.3 Anaplasma marginale species-specific PCR of the msp1a gene 69 
5.2.4 Anaplasma marginale species-specific PCR of the msp4 gene 69 
ix 
5.2.5 DNA sequence alignment and phylogenetic analysis 72 
5.3 Results 73 
5.3.1 Phylogenetic analysis of A. marginale isolates using msp1a gene 74 
5.3.1.1 MSP1 a sequence analysis 74 
5.3.2 Phylogenetic analysis using msp4 gene sequences 79 
5.3.3 Phylogenetic analysis of msp4 gene using neighbor-joining phylogenetic tree 79 
5.3.4 Phylogenetic analysis of msp4 gene using maximum-likehood phylogenetic 83 
tree 
5.4 Discussion 86 
5.4.1 Phylogenetic analysis of A. marginale using msp1a gene 87 
5.4.2 Phylogenetic analysis of A. marginale using msp4 gene 90 
6. GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS 
6.1 General discussion 92 
6.2 Conclusions 93 
6.3 Recommendations 95 
REFERENCES 96 
x 
LIST OF FIGURES 
Figure 1: Bovine erythrocytes infected with A. marginale. (A) Inclusion bodies located 8 
at the periphery of the erythrocyte in a stained blood smear. (B) Electron 
micrograph of A. margina/e inclusion that contains four organisms (Zivkovic, 
2010). 
Figure 2: The life cycle of A. marginale. The cycle is modified from Kocan (1999), to 11 
include the replication of the rickettsia in endothelial cells (Carreno et al. , 
2007) and Rhipicephalus microp/us ticks present in Mexico (Rodriguez et al. , 
2009). 
Figure 3: Gel image of 1%  agarose gel electrophoresis of PCR amplification products 39 
obtained from A. marginale isolates from Western Cape Province in South 
Africa using 1733F and 2973R primers (Lew et al. , 2002). 
Figure 4: Prevalence of A. marginale by province in South Africa. 40 
Figure 5: Map of South African areas included in the study. Map of South Africa 47 
showing the provinces that were included in the study (Limpopo, 
Mpumalanga, North West, Gauteng, KwaZulu-Natal , Eastern Cape, Western 
Cape, and Northern Cape). Endemic, epidemic, and A. marginale-free areas 
are coloured (data collected from de Waal, 2000). The main tick species 
involved in the transmission of A. marginale in the sampled areas are 
shown: Rhipicephalus microplus, R. decoloratus, and R. evertsi evertsi (data 
collected from de Waal, 2000). 
Figure 6: Newly reported sequences of MSP1 a tandem repeats. The one-letter amino 54 
acid code was used to depict msp1a repeat sequences. Dots indicate 
identical amino acids, and gaps indicate deletion/insertions. The ID of each 
repeat form was assigned previously in Cabezas-Cruz et al. (2013). Tandem 
xi 
repeat A was used as a model for amino acid comparison. 
Figure 7: Correlation between msp1a genetic diversity and A. marginale prevalence in 59 
South Africa. The prevalences of anaplasmosis in different South African 
provinces were plotted against the average of genetic diversity index (GDI). 
GDI . was calculated for each strain as the number of different tandem 
repeats divided by the total number of tandem repeats. A polynomial 
correlation was found between these 2 parameters with R2 = 0.7 6. 
Provinces with 100% prevalence (Gauteng, Eastern Cape, and 
Mpumalanga) are in the range of 0.82-0.87 GDI while regions having less 
than 100% prevalence are out of this range. 
Figure 8: Reconstruction of ancestral amino acid sequence and amino acid positions 63 
under positive and negative selection. The reconstruction of the ancestral 
state (TR 4: Tandem repeat 4, sequence marked with *) of the new tandem 
repeats found in South Africa (Figure 2) was performed using 3 
reconstruction methods, namely: joint, marginal, and sample (see 'Materials 
and methods' for details). Positions that evolve under negative (-) and 
positive (+) selection are shown (see Table 4). Amino acid at position 20 is 
indicated (arrow). The residues of the immunodominant B-cell epitope 
(Garcia-Garcia et al. , 2004) (first box) and the neutralization-sensitive 
epitope (Palmer et al. , 1987; Allred et al., 1990) (second box) are also 
shown. 
Figure 9: MSP1 a amino acid variability, composition, and low-variable peptides. The 65 
figure shows the amino acid variability and composition among the tandem 
repeats in 3 different countries: Venezuela (A), USA (B), and South Africa 
(C). Venezuela shows a high proportion of variable/conserved sites and a 
high average of amino acid variability while South Africa and the USA show 
middle and lower values, respectively. Different colours in columns depict 
different biochemical properties in the amino acid composition: negative 
xii 
(green), positive (red), uncharged-polar (beige), and non-polar (blue); 
proportion of deleted positions is shown in yellow. Consensus sequences of 
low-variable peptides are shown (*) for the USA and South Africa. The region 
of the immunodominant B-cell epitope from A. marginale (Garcia-Garcia et 
al. , 2004) is boxed in the low-variable peptide from South Africa. (For 
interpretation of the references to colour in this figure legend, the reader is 
referred to the web version of this article.) 
Figure 10: Gel image of 1%  agarose gel electrophoresis of PCR amplification products 71 
obtained from A. marginale isolates from Western Cape Province in South 
Africa using MSP45F (de la Fuente et a/. ,2007) and MSP45R (Oberle and 
Barbet, 1993). Lane M: GeneRuler™ I Kb DNA ladder, ready-to-use; Lane 1-
10 indicate positive results and lane 11 indicate negative control and lane 12 
positive control. 
Figure 11: Proportional identity between tandem repeat in position R1 and R1 +n. 75 
Figure 12: Maximum Likelihood phylogenetic tree based on the msp1a from the strains 78 
present in KwaZulu-Natal and North West provinces. The tree demonstrates 
that the strain A. marginale KZN-K (red dot) falls in the cluster from North 
West sequences (blue circle). Bootstrap values are shown as percentage in 
the internal branch. Only bootstrap values higher than 50 per cent are 
shown. NJ phylogenetic analysis provided similar results. 
Figure 13a: Neighbor-joining phylogenetic tree of A. marginale msp4 gene sequences 81 
from strains identified in cattle in South Africa. The phylogenetic tree was 
implemented in the MEGA5 (Tamura et al., 2011 ). Bootstrap analysis was 
conducted with 1000 replicates. The GenBank accession numbers of the 
respective sequences used for the phylogenetic analysis are indicated at the 
beginning of the name of each sequence. 
xiii 
Figure 13b: Neighbor-joining phylogenetic tree of A. marginale msp4 gene sequences 82 
from strains identified in cattle in South Africa represented with a green dot 
and blue dot representing an outgroups (A ovis and A. phagocytophilum) 
including isolates from West, East and North Africa , North and South 
America, Europe, Asia and Australia. The phylogenetic tree was 
implemented in the MEGA5 (Tamura et al., 2011 ). Bootstrap analysis was 
conducted with 1000 replicates. The Gen Bank accession numbers of the 
respective sequences used for the phylogenetic analysis are indicated at the 
beginning of the name of each sequence. Only bootstrap values higher than 
50% are shown. 
Figure 14a: Maximum-likelihood phylogenetic tree of A. marginale msp4 gene 84 
sequences from strains identified in cattle in South Africa. The phylogenetic 
tree was implemented in the MEGA5 (Tamura et al., 2011 ). Bootstrap 
analysis was conducted with 1000 replicates. The Gen Bank accession 
numbers of the respective sequences used for the phylogenetic analysis are 
indicated at the beginning of the name of each sequence. 
Figure 14b: Maximum-likelihood phylogenetic tree of A. marginale msp4 gene 85 
sequences from strains identified in cattle in South Africa represented with a 
green dot and blue dot representing an outgroup including isolates from 
West, East and North Africa, North and South America, Europe, Asia and 
Australia. The phylogenetic tree was implemented in the MEGA5 (Tamura et 
al. , 2011 ). Bootstrap analysis was conducted with 1000 replicates. The 
GenBank accession numbers of the respective sequences used for the 
phylogenetic analysis are indicated at the beginning of the name of each 
sequence. Only bootstrap values higher than 50% are shown. 
xiv 
LIST OF TABLES 
Table 1: Ticks and the pathogens they transmit in cattle in South African provinces 6 
Table 2: Estimated costs of tick and tick-borne diseases to cattle production 13 
Table 3: Oligonucleotide sequences of primers used in this study 36 
Table 4: Sampling sites and their coordinates in South African provinces 48 
Table 5: Observed prevalences of A. marginale in different provinces of South Africa 51 
Table 6: Anaplasma marginale strains and msp1a tandem repeats organization 57 
Table 7: Sites that evolved under positive and negative selection in the new tandem 62 
repeats from South Africa 
Table 8: Oligonucleotide sequence of primers used in this study and their references 68 
Table 9: Anaplasma marginale strains and putative 20 structures of MSP1a tandem 76 
repeats 
Table 10: Differences in the A. margina/e msp4 nucleotide sequences of 80 
LP,NW,GP,KZN,WC and MP, EC isolates of South Africa 
xv 
APPENDICES 
Appendix 1: Raw data used for statistical analysis of A. marginale in cattle in provinces of 132 
South Africa 
Appendix 2: GenBank accession number of A. margina/e msp1a gene isolates and their 139 
origin in South Africa 
Appendix 3: GenBank accession number of A. marginale msp4 gene isolates and their 142 
origin in South Africa 
Appendix 4: Amplified sequences of Anaplasma spp. isolates, their origin and GenBank 146 
accession numbers 
xvi 
LIST OF ABBREVIATIONS 
oc degree Celsius 
BLAST Basic Local Alignment Search Tool 
BLASTn Basic Local Alignment Search Tool for Nucleotide 
bp base pair 
CAT Card Agglutination Test 
cELISA Competitive enzyme-linked immunosorbent assay 
CF Complement fixation test 
DNA deoxyribonucleic acid 
EC Eastern Cape 
EDTA Ethylenediamine tetra -acetic acid 
GDI Genetic diversity index 
GP Gauteng Province 
IFA Indirect fluorescent antibody 
Kb Kilo base 
kDa Kilodalton 
KZN KwaZulu-Natal 
LP Limpopo Province 
MAb Monoclonal antibody 
mM millimolar 
ml milliliter 
MP Mpumalanga 
MSP Major Surface Protein 
NC Northern Cape 
NCBI National Center for Biotechnology Information 
ng nanogram 
ng/µI nanogram per microliter 
NW North -West 
PCR polymerase chain reaction 
TBD Tick-borne diseases 
xvii 
UV ultraviolet 
WC Western Cape 
µg microgram(s) 
µm micrometer 
µM micromolar 
xviii 
RESEARCH OUTPUTS 
Publications 
Awelani M. Mutshembele. Alejandro Cabezas-Cruz, Moses S. Mtshali, Oriel , M. M. Thekisoe, Ruth 
C. Galindo and Jose de la Fuente. 2014. Epidemiology and evolution of the genetic variability of 
Anaplasma marginale in South Africa. T icks and Tick -borne diseases, Volume 5, Issue 6, 624-631 . 
Conferences 
Awelani M. Mutshembele, A. Cabezas-Cruz, M. S. Mtshali, 0 . M. M. Thekisoe, R. C. Galindo, J de 
la Fuente. Epidemiology and evolution of the genetic variability of Anaplasma marginale in South 
Africa. 81h International Ticks and Tick-Borne Pathogens and Biennial Social Tropical Veterinary 
Medicine (TTP-STVM) Conference. Cape Town, South Africa. 24-29 August 2014. 
Awelani M. Mutshembele, M. S. Mtshali, 0 . M. M. Thekisoe, R. C. Galindo, A. Cabezas-Cruz, J de la 
Fuente. Molecular prevalence, genetic diversity and phylogenetic analysis of Anaplasma marginale 
isolates in cattle in South Africa. 42"d PARSA Conference hosted by the North West University, 
Stonehenge in Africa, Parys, Free State, 22-24 September 2013. 
Seminars and Symposia 
Awelani M. Mutshembele, M.S. Mtshali and 0 . M. Thekisoe. Molecular detection, genetic diversity 
and phylogenetic analysis of Anaplasma marginale isolates in cattle in South Africa. Postgraduate 
seminar day, University of the Free State- QwaQwa campus. 13-14 November 2013. 
Awelani M. Mutshembele, A. Cabezas-Cruz, M. S. Mtshali, 0 . M. M. Thekisoe, R. C. Galindo, J de la 
Fuente. Structura l and epidemiological analysis of Anaplasma marginale using the major surface 
protein 1a  in South Africa. 4 th National Zoological Gardens of South Africa (NZG) Research 
Symposium, November 2013. 
xix 
Awelani M. Mutshembele, Moses S. Mtshali, Oriel M. M. Thekisoe. The genetic diversity and 
phylogenetic analysis of Anap/asma marginale strains in cattle in South Africa. The Zoology 
Department Seminar, Faculty of Zoology and Entomology, University of the Free State, Bloemfontein 
Campus, November 2012. 
Awelani M. Mutshembele, Moses S. Mtshali, Oriel M. M. Thekisoe. The genetic diversity and 
phylogenetic analysis of Anaplasma marginale strains in cattle in South Africa. 3rd National Zoological 
Gardens of South Africa (NZG) Research Symposium, November 2012. 
Awelani M. Mutshembele, Moses S. Mtshali, Oriel M. M. Thekisoe. Analysis of phylogeographic 
relationships of Anaplasma marginale from South African isolates using msp4 gene sequences. The 
Zoology Department Seminar, Faculty of Zoology and Entomology, QwaQwa campus. November 
2011 . 
xx 
ABSTRACT 
Bovine anaplasmosis caused by Anap/asma marginale is endemic in South Africa. This 
endemicity is due to presence of tick vectors that transmit A. marginale the causal agent 
of the disease and the high seroprevalence in Limpopo, Free State and North West 
provinces. To date, the genetic diversity of A. marginale isolates infecting cattle in all 
South African provinces, except Free State, are generally unknown. Recently, vaccines 
based on the A. marginale major surface protein 1a  (MSP1 a) has been proposed as a 
strategy for controlling bovine anaplasmosis. However, characterization of genetic 
diversities of the A. marginale isolates in these regions is still needed before this protein 
can be used for vaccine development. Therefore, the aim of this study was to determine 
the prevalence, genetic diversity and phylogenetic relationship of A. marginale infecting 
cattle in all South African provinces except the Free State. 
A total of 280 whole blood samples were collected from cattle in all provinces with 
exception of the Free State. Twenty six districts and municipalities were included in this 
sampling. Anaplasma marginale genomic DNA was then extracted from the blood 
sample using ZR Genomic DNA 1M Tissue Miniprep (Zymo Research, CA, USA). A 
polymerase chain reaction (PCR) was done with primers targeting msp1a and msp4 
genes and the PCR products were sequenced using genetic analyser (ABI , Life 
technologies, CA, USA). The generated sequences were analysed by bioinformatics 
and their phylogeny as well as genetic diversity index (GDI) was determined based on 
the sequences of msp1a and msp4 genes. 
xxi 
Overall , the prevalence of A. marginale infection in cattle was 76% in all provinces 
except for Northern Cape Province where the prevalence was zero. The prevalence per 
province was as follows: Eastern Cape 19.1% , Gauteng 9.6%, KwaZulu-Natal 23.0%, 
Limpopo 15.3%, Mpumalanga 10.1%, North West 12.4% and Western Cape 10.5%. 
The msp1 a revealed genetic variability with regions of different types of tandem repeats. 
Some repeats were conserved amongst the A. marginale strains and revealed low 
variable peptides in the MSP1 a tandem repeats. A polynomial correlation (R2=0.7 6) 
was observed between the GDI and anaplasmosis prevalence per province. 
Interestingly, provinces with the highest prevalence were not the ones with highest or 
lowest GDI. 
The analysis of msp4 gene sequences, which provided evolutionary information about 
geographically distinct A. marginale strains, was used in the present study for 
phylogenetic analysis of samples from Limpopo (LP), Mpumalanga (MP), North West 
(NW), Gauteng (GP), KwaZulu-Natal (KZN), Eastern Cape (EC) and Western Cape 
(WC) provinces of South Africa. Two clades were observed which consisted of first 
clade (LP, NW, GP, KZN and WC) and second clade (MP and EC) isolates. 
In addition when DNA sequence variation of msp4 gene was analysed in combination 
with isolates from other countries outside South Africa , important phylogeographic 
information was observed. The South African strains had 100% identity with isolates 
from Kenya, Zimbabwe and Australia. Good representation of the Southern and 
Northern Hemispheres was observed and demonstrated that the msp4 gene was a 
good phylogeographic marker. These results indicated that A. marginale is widespread 
in South Africa, and suggested that the analysis of msp1a and msp4 gene sequences 
provided an understanding of the phylogeny and epidemiology of A. marginale in South 
Africa. 
xx ii 
CHAPTER 1 
INTRODUCTION AND LITERATURE REVIEW 
1.1 Historical background 
The genus Anaplasma was established in 1910 by Sir Arnold Theiler who first described 
the "marginal points" in stained erythrocytes of sick cattle as the causative agents of a 
specific disease (Theiler, 191 O; 1911 ). These erythrocytic inclusions had been seen 
frequently in red blood cells of anemic cattle often in those suffering from babesiosis 
(piroplasmosis). The acute phase of the disease is characterized by weight loss, fever, 
abortion, lowered milk production and often death (Kuttler, 1984). 
1.2 Classification of Anaplasma marginale 
Bovine anaplasmosis is a tick-borne rickettsial disease caused by the hemoparasite 
Anaplasma marginale (Aubry and Geale, 2011 ). Anaplasma marginale is classified 
within the Order Rickettsiales which was recently reorganized into two fam ilies, 
Anaplasmataceae and Rickettsiaceae based on genetic analysis of 16S rRNA, groELS 
and surface protein genes (Dumler et al. , 2001 ). Members of the family 
Anaplasmataceae are obligate intracellular organisms found exclusively within 
membrane-bound vacuoles in the host cell cytoplasm. The genus Anaplasma includes 
three species that infect ruminants, namely A. marginale, A. centrale and A. ovis 
(Dumler et al., 2001 ), also included within this genus is A. phagocytophilum, agent of 
human granulytic ehrlichiosis [HEG], A. bovis and A. platys (Kocan et al., 2010). 
Anaplasma marginale is a gram- negative rickettsia which is endemic in tropical and 
subtropical areas throughout the world (Decaro et al. , 2008; Howden and Geale, 201 O; 
Kocan et al. , 201 O; Aubry and Geale, 2011 ). Bovine anaplasmosis is a major constra int 
to cattle production in many countries (Kocan et al., 2010). 
1 
In order to persist in nature, A. marginale infects the mammalian host which usually 
remains persistently infected (Kieser et al., 1990; Eriks et al., 1993) serving as a 
reservoir for infection of ticks or mechanical transmission by the transfer of blood from 
infected to susceptible cattle (Kocan et al. , 1992; Ge et al., 1996; Futse et al., 2003). 
Taxonomic Classification of Anaplasma infecting ruminants (Dumler et al. , 2001 ): 
Kingdom: Bacteria 
Phylum: Proteobacteria 
Class: Alphaproteobacteria 
Order: Rickettsiales 
Family: Anaplasmataceae 
Genus: Anap lasma 
Species: A. marginale 
A. centrale 
A. bovis 
A. ovis 
A. phagocytophilum 
A. platys 
1.3 Epidemiology 
Bovine anaplasmosis occurs in tropical and subtropical regions of the world and the 
disease is a major constraint to cattle production in many countries. Anaplasma 
marginale, in contrast to A. phagocytophilum, is quite host specific, infecting only 
ruminants and causing disease primarily in cattle. In the U.S.A. it is enzootic throughout 
the southern Atlantic states, Gulf Coast states, and several of the Midwestern and 
Western states (Kocan et al., 2010). 
2 
However, it has been reported in almost every state in the U.S.A. This increasingly wide 
distribution likely resulted from transport of carrier cattle with subsequent mechanical or 
biological transmission from asymptomatic persistently infected cattle to susceptible 
ones (Kocan et al. , 2010). 
It is also endemic in Mexico, Central and South America, as well as in the Caribbean 
Islands. It is enzootic in all Latin American countries, with the exception of desert areas 
and certain mountain ranges, like the Andes. The seroprevalence rates of A. marginale 
vary widely among countries in the America and the variability of these rates contributes 
to the development of geographically stable enzootic regions (Kocan et al. , 2010). 
In Europe, A. marginale is found mainly in Mediterranean countries with infections 
having been described in cattle and assorted wildlife species. It is also endemic in 
regions of Asia and Africa. The distribution of anaplasmosis may be expected to 
continue to change, in part as a result of global warming, which may influence the 
movement of the tick hosts. An example of the validity of such a prediction is a 
confirmed diagnosis of anaplasmosis in a bison herd in Saskatchewan, Canada, during 
the summer of 2000. The first reported outbreak of anaplasmosis in Canada occurred in 
1971 , but this outbreak resulted from mechanical transmission from imported carrier 
cattle to local ones (Kocan et al. , 2010). 
In South Africa only one study has been conducted to genetically characterize the 
geographical strains (Mtshali et al., 2007). The results presented by the latter authors 
indicated the presence of a common genotype between South African, American and 
European strains of A. marginale. However the study focused only on blood samples of 
cattle collected from Free State province, so there is a need of further documentation of 
prevalence and genetic diversity of A. marginale strains in cattle of South African origin 
in order to design epidemiological and control strategies. 
3 
1.4 Distribution of ticks infesting cattle in South Africa 
Several species of ticks are found in different geographical areas in South Africa. The 
type of vegetation, humidity and temperature influences their distribution. Studies in the 
eastern Free State revealed that ticks affect mainly cattle of small farmers and the main 
tick species in the area are Rhipicephalus decoloratus (53.1% ), R. evertsi evertsi 
(44.7%), R. fol/is (1 .0%), R. gertrudae (0.7%) and R. warburtoni (0.4%). In the south 
west region of the same province, Fourie and Horak (1991) observed that Amblyomma 
marmoreum, Hyalomma marginatum rufipes and H. truncatum were the predominant 
species. A second study by Fourie et al. (1996) in the south west region revealed that 
lxodes rubicundus and H. m. rufipes were the most prevalent tick species (Marufu, 
2008). 
In KwaZulu-Natal, Baker et al. (1989) observed that R. decoloratus, R. appendiculatus 
and R. evertsi evertsi were the most prevalent tick species of cattle raised on 
commercial farms. H. m. rufipes , R. appendiculatus and R. evertsi evertsi were the most 
numerous species in the Limpopo Province also present were R. microplus, R. 
decoloratus, A. hebraeum, H. truncatum and R. simus. Bryson et al. (2002) noted that 
the adults of A. hebraeum, R. appendiculatus and R. evertsi evertsi were the most 
numerous tick species in North West Province. In Mpumalanga, R. decoloratus 
constituted more than 75% of the total tick population. In a five year survey conducted in 
the Eastern Cape Province, R. decoloratus, A. hebraeum, R. appendiculatus and R. 
evertsi evertsi were found to be the most common tick species infesting cattle (Marufu, 
2008). 
4 
In contrast to these early findings, Horak (1999) observed A. hebraeum, Haemaphysalis 
silacea, R. appendiculatus and R. glabroscutatum to be most prevalent tick species on 
yearling commercial cattle on Valley Bushveld. Muchenje et al. (2008) in an on station 
study comparing tick loads on Nguni, Angus and Bonsmara steers grazing on sweet 
rangeland revealed that R. decoloratus, A. hebraeum, R. evertsi evertsi and Hyalomma 
species were the most common tick infestations. With the exception of Hyalomma 
species, the same tick species composition observed by Muchenje et al. (2008), was 
found to infest cattle and goats in the communal areas of the Eastern Cape (Nyangiwe 
and Horak, 2007). 
Many studies have focused on cattle in the commercial farming system and it is 
apparent that cattle management and tick control in this farming sector will differ 
considerably to that in communal farming areas (Bryson et al., 2002). Few studies have 
focused on cattle kept by resource-poor farmers in communal areas. These studies 
however, did not compare the prevalence of the parasites in different breeds kept under 
communal farmer management and across different rangelands types. Other studies 
have focused on comparing tick loads in indigenous and exotic beef breeds under 
controlled conditions (Norval et al. , 1996; Muchenje et al. , 2008). No studies have 
focused on the comparison of tick loads in the indigenous and nondescript cattle under 
communal grazing management. Information on the tick loads of cattle can be used in 
conjunction with sere-diagnostic methods to estimate and compare the level of 
resistance of different cattle breeds to ticks (Wambura et al., 1998; Mattioli et al., 2000). 
Tick distribution and occurrence differs with geographic distribution and vegetation type 
(Mtshali et al. , 2004). No efforts have been made to compare tick loads in cattle on 
sweet and sour rangelands. Sour rangeland occurs in areas with high water supply and 
denser vegetation cover (Ellery et al., 1995) and it is more likely to have higher 
prevalence of ticks than the sweet rangeland which occurs in areas with low water 
supply and sparse vegetation cover. Comparing the prevalence of ticks in different 
rangeland types assists policy makers to design appropriate control programmes for 
each particular rangeland type (Marufu, 2008). 
5 
The most common tick species and the disease that they transmit to cattle in South 
Africa are shown in Table1 (Marufu, 2008). 
Table 1: Ticks and the pathogens they transmit to cattle in South African 
provinces 
Tick species Pathogens transmitted Geographical 
distribution (Provinces) 
Rhipicephalus (Boophilus) Anaplasma marginale, Limpopo, Mpumalanga, 
spp Gauteng, North West, 
Babesia bigemina, KwaZulu-Natal , Eastern 
B. bovis and Northern Free State, 
Eastern Cape and Coastal 
~rip ~ Sou~ern and 
South-western Cape 
R. appendiculatus Theileria parva Limpopo, Mpumalanga, 
KwaZulu-Natal, Western 
T. annulata Cape 
T. taurotragi 
T. mutans 
T. velifera 
R. evertsi evertsi A. marginale Eastern half of Southern 
Africa, South-western 
Cape 
Hylomma spp A. marginale Karoo, Southern Africa 
except coastal Eastern 
Cape, KwaZulu-Natal, 
Eastern Southern and 
South-western Cape 
Amblyomma hebraeum Ehrlichia (Cowdria) Limpopo, Mpumalanga, 
ruminantium KwaZulu-Natal and 
Eastern and Southern 
T. mutans South-western Cape 
Sources: Horak et al. , 1991 ; Walker, 1991 ; Norval et al., 1992; Coetzer et al. , 1994. 
6 
1.5 Pathogenesis of A. marginale 
Anaplasma marginale is known to infect only mature, circulating erythrocytes of 
domestic and wild ruminants in vivo (Figure 1A). However, recently in vitro and in vivo 
studies indicated that it also infects endothelial cells, which may have implications for 
both pathogenesis and immune mechanisms. A. marginale enters erythrocytes by 
endocytosis and resides in the membrane-bound vacuole, where it divides by binary 
fission . The membrane vacuole is derived from erythrocyte membrane and contains 4 to 
8 organisms (Figure 1B ). In the acute infection as much as 70% of erythrocytes may 
become infected (Zivkovic, 2010). 
During high rickettsiemias (bacteremia) multiple infections of individual erythrocytes are 
common. The incubation period varies with the infective dose and ranges from 7 to 60 
days, with an average of 28 days. A. marginale leaves the host cell without disrupting it. 
Erythrocytes that are physically or chemically altered during the course of the disease 
are recognized by bovine reticulo-endothelial cells and phagocytized, which will result in 
the development of mild to severe anemia and icterus, without hemoglobinemia and 
hemoglobinuria (Zivkovic, 2010). 
The acute phase of the disease may also include symptoms such as high fever, 
dramatic weight loss, abortion, lethargy and often death in animals older than 2 years. 
Calves less than one year of age develop a relatively mild form of disease. Importantly, 
cattle that survive acute disease develop a lifelong persistent infection and serve as 
reservoirs for transmission to new susceptible hosts. Persistence is characterized by 
sequential rickettsemic cycles, occurring at approximately 5 week intervals, with peaks 
at 106 bacteria /ml of blood followed by a rapid decline when rickettsemia is controlled 
by a specific immune response. Persistently infected cattle have a lifelong immunity and 
are resistant to clinical onset of the disease on challenge exposure (Zivkovic, 2010). 
7 
Figure 1: Bovine erythrocytes infected with A. marginale. (A) Inclusion bodies located 
at the periphery of the erythrocyte in a stained blood smear. (B) Electron micrograph of 
A. marginale inclusion that contains four organisms (Zivkovic, 2010). 
8 
1.6 Transmission 
Transmission of A. marginale can be both mechanical by biting flies or blood-
contaminated fomites and biologically by ticks. Mechanical transmission frequently 
occurs via blood-contaminated fomites , including needles, dehorning saws, nose tongs, 
tattooing instruments, ear-tagging devices, and castration instruments. Mechanical 
transmission by arthropods has been reported for bloodsucking diptera of the genera 
Tabanus, Stomoxys, and mosquitoes. This form of mechanical transmission is 
considered to be the major route of dissemination of A. marginale in areas of Central 
and South America and Africa where tick vectors do not occur and where Rhipicephalus 
(Boophilus) microplus, the tropical cattle tick, does not appear to be a biological vector 
of A. marginale. In areas of the United States where geographic isolates of A. marginale 
are not infective for ticks or where ticks have been eradicated by fire ants, mechanical 
transmission appears to be the major mode of A. marginale transmission (reviewed by 
Kocan et al. , 2010). 
In addition to mechanical and biological transmission, A. marginale can be transmitted 
from cow to calf transplacentally during gestation. For example, a 15.6% prevalence 
rate of in utero transmission of Anaplasma infections was reported in South Africa 
Transplacental transmission of anaplasmosis may therefore contribute to the 
epidemiology of this disease in some regions. This obligate intracellular pathogen can 
be transmitted biological by ticks, and approximately 20 species of ticks have been 
shown experimentally to transmit A. marginale worldwide. Tick transmission can occur 
from stage to stage (transstadial) or within a stage (intrastadial), while transovarial 
transmission from one tick generation to the next does not appear to occur. lnterstadial 
transmission of A. marginale has been demonstrated by the three-host ticks 
Dermacentor andersoni and D. variabilis in the United States and by R. simus in South 
Africa. The one-host tick R. annulatus transmits A. marginale in Israel, Central America, 
South America, and Mexico (reviewed by Kocan et al., 2010). 
9 
lntrastadial transmission of A. marginale is effected by male ticks. Recent studies have 
demonstrated that male Dermacentor ticks may play an important role in the biological 
transmission of A. marginale because they become persistently infected with A. 
marginale and can transmit the parasite repeatedly when they transfer among cattle. 
Male ticks therefore also serve as reservoirs of A. marginale along with persistently 
infected cattle. Transmission of A. marginale by male ticks may be an important 
mechanism of transmission by one-host ticks, including Rhipicepha/us (Boophilus) spp. 
and D. albipictus. However, it was shown recently that the co-feeding of adult 
Dermacentor spp. does not appear to influence the dynamics of A. marginale 
transmission (reviewed by Kocan et al., 2010). 
1.7  Life cycle of A. marginale 
The life cycle of A. marginale in ticks is complex and well-coordinated with the tick 
feeding cycle (Figure 2). Infected erythrocytes are ingested by ticks with a blood meal 
and the first sites of infection are gut and malpighian tubule cells. During the 
subsequent feeding many other tissues, including salivary glands, become infected from 
where A. marginale can be transmitted to the vertebrate host. At each site of infection 
two stages of A. marginale occur within a membrane bound vacuole in the tick cell 
cytoplasm (Zivkovic, 2010). 
The first form seen within A. marginale colonies is the reticulated (vegetative) form, 
which divides by binary fission and results in formation of large colonies containing 
hundreds of organisms. The reticulated forms are then transformed into dense forms, 
which are the infective form and can survive for a short time outside of cells. Cattle 
become infected when the dense form is transmitted during tick feeding via the salivary 
glands. Ticks are able to acquire infection after feeding on persistently infected animals 
with a very low level of rickettsemia. Moreover, once ticks acquire the infection the 
biological replication of the organism with in the ticks makes up for the initial low infective 
dose (Zivkovic, 2010). 
10 
ee In ction o 
dense forms 
A marginale in the bovine 
Endothelial Cells Rhipicephalus 
Erythrocytes spp 
Replication in sevenl 
• tissues within the tick •• • .. .. ..  •  I 
• 
Feed acquisition of 
infected erythrocvtes 
/ 
Rhipicephalus 
spp 
rigure 2: The life cycle of A marginale. The cycle is modified from Kocan (1999), to include the replication 
l~ the rickettsia in endothelial cells (Carreno et al., 2007) and Rhipicephalus microplus ticks present in 
rexico (Rodriguez et al., 2009). 
I 
11 
1.8 Geographic distribution and economic importance of anaplasmosis 
The distribution of anaplasmosis may be expected to continue to change in part as a 
result of global warming, which may influence the movement of the tick hosts (Jonsson 
and Reid , 2000. Concerns regarding the transmission of infectious agents between 
wildlife and domestic livestock are increasing especially in areas where free-ranging 
wildlife and cattle share common grazing grounds (Chomel et al., 1994). Bovine 
anaplasmosis is endemic almost anywhere the world from the Far East to Australia, 
Africa, Europe and the Americas (Wen et al. , 2002; Ziam and Benaouf, 2004; Naranjo et 
al. , 2006; Videtto et al., 2006; Stevens et al. , 2007). Bovine anaplasmosis causes 
important economic loss in most countries, mainly due to the high morbidity and 
mortality in susceptible cattle herds (Marufu, 2014). 
Losses due to anaplasmosis are measured through several parameters: low weight 
gain, reduction in milk production, abortion, the cost of anaplasmosis treatments, and 
mortality. However, few controlled studies have been carried out to determine the exact 
annual loss caused by anaplasmosis. The most important economic constraint of 
anaplasmosis to cattle production in the tropics is on public or private programs for 
genetic improvement of cattle. Imported Bos Taurus cattle brought from temperate 
nations to the tropics for breed improvement are highly susceptible to tick-borne 
diseases (TBD), and often do not survive to become part of planned production 
programs. This constraint is a notable reality for programs for the improvement of cattle 
in most Latin American countries (Marufu, 2014). 
The lack of accurate data on the epidemiology of ticks and TBD makes it difficult to 
determine their impact. The complexity of determining the direct and indirect economic 
impact of ticks and TBD, and their control is reflected in the fact that only rough 
estimates are available for the cost of some of the components. Table 2 shows the 
estimated costs of ticks and TBD to cattle production in different countries. Although a 
fairly crude estimate, these values may help to comprehend the importance of ticks and 
TBD in cattle. These estimates, however, expose the need for more studies on the 
determination of the economic impact of ticks and TBD in the cattle industry especially 
in the developing world (Marufu, 2014). 
12 
Table 2: Estimated costs of tick and tick-borne diseases to cattle production 
Country Costs (US$) Reference 
Global 13-18 billion de Castro (1997) 
Southern 31.6 million Minjauw et al., (1998) 
Africa 
Australia 4.09 million Jonsson et al., (2001) 
India 498.7 million Minjauw and Mcleod (2003) 
Australia 170 - 200 million Playford (2005), Sackett et al., (2006) 
Brazil 800 million Martinez et al., (2006) 
Losses are partly due to the direct effects of ticks on cattle, such as damaged hides and 
skins, anaemia, reduced body weight gains and milk yield, tick toxicoses and mortalities 
(Gates and Wescott, 2000; Turton, 2001 ; Mtshali et al. , 2004; Kaufman et al. , 2006). 
The damage caused by tick bites also diminishes the value of skins and hides for the 
manufacture of leather. Ticks with long mouth parts may induce abscesses because of 
secondary bacterial infections. Depending on the site of infestation, these abscesses 
can lead to lameness or mastitis resulting in the drop in milk production and subsequent 
increase in calf mortalities (Marufu, 2014). A large component of the economic cost of 
ticks in cattle is the appl ication of control measures to reduce infestations (de Castro, 
1997; Porto Neto et al. , 2011 ). Conventional tick control is based on the appl ication of 
acaricides. The practice of intensive tick control spread rapidly throughout Africa 
following the introduction of imported cattle breeds and, in South Africa, it was enforced 
through legislation. There are few global reports on the costs involved in tick control and 
TBD treatments (Marufu, 2014). 
13 
Financial analysis revealed that spraying cattle with acaricide twice a week yields a 
return of 244% and maximised financial benefits to the farmer (Mukhebi et al. , 1989). 
Jonsson et al., (2001 ), however, estimated the total costs of tick control to contribute up 
to 49% of the total costs of ticks and TBD on the dairy industry in Australia. 
Expenditures for tick control were estimated at US$ 8.43, 13.62 and 21 .09 per animal 
per year for plunge dipping, hand spraying and pour-on, respectively (D'haese et al., 
1999). The mean annual cost of ticks and TBD control per animal in pastoral and ranch 
herds was estimated to be US$4.54 (Ocaido et al., 2009). The development of new 
acaricides is also a lengthy and costly process leading to increasing cost of the newer 
products. Regular dipping has also led to the loss of resistance to ticks and enzootic 
stability to TBD. Significant losses also arise indirectly due to the important role of ticks 
in the transmission of TBD (Marufu, 2014). 
1.9 Diagnosis of anaplasmosis 
The diagnostic assays (excluding cl inical findings) used to identify A. marginale can be 
classified as microscopic, serologic assays such as Indirect fluorescent antibody (IFA), 
complement fixation (CF) test, capillary, agglutination assay, card agglutination test 
(CAT), indirect fluorescent antibody (IFA) test, as well as various enzyme linked 
immunosorbent assays (ELISA) such as a cELISA, indirect ELISA and dot ELISA. The 
preferred serological tests are cELISA and CAT and molecular diagnostic assay using 
polymerase chain reaction (PCR) of whole blood samples (revised in Aubrey and Geale, 
2011 ). However, the use of microscopy requires careful examination in cases of low 
level of parasitemia and A. marginale appear as dense, rounded and deeply stained 
intraerythrocytic bodies, approximately 0.3- 1.0 µm in diameter. Most of these bodies 
are located on or near the margin of the erythrocyte. This feature distinguishes A. 
marginale from A. centrale, as in the latter most of the organisms have a more central 
location in the erythrocyte. It can be difficult to differentiate A. marginale from A. 
centrale in a stained smear, particularly with low levels of rickettsaemia (OIE Terrestrial 
Manual 2012). 
14 
Anaplasma infections usually persist for the life of the animal. However, except for 
occasional small recrudescences, Anaplasma cannot readily be detected in blood 
smears after acute rickettsaemia. Thus, a number of serological tests have been 
developed with the aim of detecting persistently infected animals. A feature of the 
serological diagnosis of anaplasmosis is the highly variable results with regard to both 
sensitivity and specificity reported for many of the tests from different laboratories. This 
is due at least in part to inadequate evaluation of the tests using significant numbers of 
known positive and negative animals. Importantly, the capacity of several assays to 
detect known infections of long-standing duration has been inadequately addressed. An 
exception is cELISA, which has been validated using true positive and negative animals 
defined by nested PCR (Torioni De Echaide et al. , 1998), and the card agglutination 
assay, for which relative sensitivity and specificity in comparison with the cELISA has 
been evaluated (Molloy et al., 1999). 
It should be noted that there is a high degree of cross-reactivity between A. marginale 
and A. centrale, as well as cross-reactivity with both A, phagocytophilum and Ehrlichia 
spp. in serological tests (Dreher et al. , 2005; Al-Adhami et al., 2011 ). While the infecting 
species can sometimes be identified using antigens from homologous and heterologous 
species, equivocal results are obtained on many occasions (OIE Terrestrial Manual 
201 2). 
1.9.1 Competetive enzyme -linked immunosorbent assay 
A cELISA using a recombinant antigen termed rMSP5 and MSP5-specific monoclonal 
antibody (MAb) have proven very sensitive and specific for detection of Anaplasma-
infected animals (Hofmann-Lehmann et al., 2004; Stik et al. , 2007; Reinbold et al. , 
2010). All A. marginale strains tested, along with A. ovis and A. centrale , express the 
MSP5 antigen and induce antibodies against the immunodominant epitope recognised 
by the MSP5-specific MAb. A recent report suggests that antibodies from cattle 
experimentally infected with A. phagocytophi/um will test positive in the cELISA (Dreher 
et al. , 2005). 
15 
However, in another study no cross-reactivity could be demonstrated, and the MAb 
used in the assay did not react with A. phagocytophilum MSP5 in direct binding assays 
(Stik et al., 2007). Recently, cross reactivity was demonstrated between A. marginale 
and Ehrlichia spp, in naturally and experimentally infected cattle (Al- Adhami et al., 
2011 ). Earlier studies had shown that the cELISA was 100% specific using 261 known 
negative sera from a non-endemic region , detecting acutely infected cattle as early as 
16 days after experimental tick or blood inoculation, and was demonstrated to detect 
cattle that have been experimentally infected as long as 6 years previously (Knowles et 
al. , 1996). In detecting persistently infected cattle from an anaplasmosis-endemic reg ion 
that were defined as true positive or negative using a nested PCR procedure, the 
rMSP5 cELISA had a sensitivity of 96% and a specificity of 95% (Torioni De Echaide et 
al., 1998). 
1.9.2 Card agglutination test 
The advantages of the card agglutination test (CAT) are that it is sensitive, may be 
undertaken either in the laboratory or in the field , and gives a result within a few 
minutes. Nonspecific reactions may be a problem, and subjectivity in interpreting assay 
reactions can result in variability in test interpretation. In addition, the CAT antigen, 
which is a suspension of A. marginale particles, can be difficult to prepare and can vary 
from batch to batch and laboratory to laboratory. Splenectomised calves are infected by 
intravenous inoculation with blood containing Anaplasma-infected erythrocytes. When 
the rickettsaemia exceeds 50%, the animal is exsanguinated, the infected erythrocytes 
are washed , lysed, and the erythrocyte ghosts and Anaplasma particles are pelleted . 
The pellets are sonicated, washed, and then resuspended in a stain solution to produce 
the antigen suspension (OIE Terrestrial Manual 2012). 
16 
1.9.3 Complement fixation test 
The complement fixation (CF) test has been used extensively for many years; however, 
it shows variable sensitivity (ranging from 20 to 60%), possibly reflecting differences in 
techniques for antigen production, and poor reproducibility . In addition , it has been 
demonstrated that the CF assay fa ils to detect a significant proportion of carrier cattle 
(Bradway et al., 2001 ). It is also uncertain as to whether or not the CF test can identify 
antibodies in acutely infected animals prior to other assays (Molloy et al. , 1999; Coetzee 
et al., 2007). Therefore, the CF test is no longer recommended as a reliable assay for 
detecting infected animals (OIE Terrestria l Manual, 2012). The use of the CF test could 
result in infected cattle being assigned a negative result, which could lead to 
introduction of persistently infected animals with false-negative resu lts into populations 
of completely na"lve cattle (Coetzee et al., 2007). 
1.9.4 Indirect fluorescent antibody test 
Other serological tests are generally preferred to the IFA test , because of the number of 
tests that can be performed daily by one operator. A serious problem encountered with 
the IFA test is non-specific fluorescence attributed to antibodies adhering to infected 
erythrocytes. Non-specific fluorescence due to antibodies adhering to infected 
erythrocytes can be reduced by washing the erythrocytes in an acidic glycine buffer 
before antigen smears are prepared (OIE Terrestrial Manual 2012). 
1.9.5 Polymerase chain reaction 
Polymerase chain reaction (PCR) has been the most commonly used method for the 
diagnosis of A. marginale infections (Lew et al., 2002, Shkap et al., 2002). Most of the 
PCR assays target the msp4 and msp1a genes for differentiating strains of A. 
marginale, which is a useful method for tracking the origin of the outbreak (Bowie et al., 
2002; Lew et al. , 2002, de la Fuente et al. , 2005d; 2007a; Mtshali et al., 2007, Cabezas-
Cruz et al., 2013; Mutshembele et al., 2014). 
17 
Polymerase chain reaction has been shown to reliably detect Anaplasma at the lowest 
levels of persistent rickettsemia (Lew et al., 2002, Shkap et al., 2002). However, other 
studies have shown that PCR fails to detect the infection of A. marginale at low levels or 
during the early stages of infection (Malad et al. , 2009). The molecular diagnosis of 
Anaplasma infection by PCR using whole blood has become readily available. Various 
genes are useful for organism identification at genus and species level. Among the most 
used are the phylogenetically reliable marker genes such as 16S RNA (Warner and 
Dawson, 1996; Wen et al., 2002), gltA (lnokuma et al., 2001 ) and groEL (Yu et al., 
2001 ). 
Polymerase chain reaction assays targeted at the Anaplasma msp4 and/or msp 1a  
genes have been used to differentiate isolates of A. marginale, which is useful to track 
the origin of an outbreak, and to differentiate between different species of Anaplasma 
such as A. marginale and A. centrale (de la Fuente et al., 2001 a; Lew et al. , 2002). 
Because msp5 expresses epitopes that are conserved among widely divergent strains 
of A. marginale, A. centrale and A. ovis (Visser et al., 1992) and even A. 
phagocytophilum (Alleman et al., 2006), a PCR assay targeted only at the msp5 gene 
would be expected to have significant specificity issues when used in populations where 
animals could be infected with other Anaplasma spp. 
Amplification of related organisms by non-specific primers has been shown to result in 
false-positive reactions. However, sequencing and sequences comparison may be a 
solution for this problem. Conversely, false-negatives may occur if extraction 
procedures fail to remove PCR inhibitors present in a blood sample or if the level of 
circulating rickettsemia falls below the level of assay. The main problem with the above 
genes is that they are conserved at species levels so strains identification becomes 
challenging or impossible using these genes. An alternative is the use of variable 
genes. The msp 1a  gene for A. marginale have become the most used tool for testing 
strains variability in these two pathogens (Palmer et al. , 2001 ; Almazan et al., 2008; 
Ruybal eta/. , 2009). 
18 
Nucleic-acid-based tests to detect A. marginale infection in carrier cattle have been 
developed although not yet fu lly val idated. The analytical sensitivity of PCR-based 
methods has been estimated at 0.0001 % infected erythrocytes, but at this level only a 
proportion of carrier cattle would be detected. A nested PCR has been used to identify 
A. marginale in carrier cattle with a capability of identifying as few as 30 infected 
erythrocytes per ml of blood, well below the lowest levels in carriers. However, nested 
PCR poses significant quality control and specificity problems for routine use (Torioni 
De Echaide et al. , 1998). 
Real-time PCR based on msp1b gene was successfully developed for detection and 
quantification of A. marginale DNA in blood of naturally infected cattle (Carelli et al. , 
2007; Decaro et al., 2008; Reinbold et al. , 2010). The assay was shown to be 
distinctively sensitive and specific as there were no cross-reactions with other 
haemoparasites of ruminants (A. centrale, A. bovis, A. phagocytophilum, B. bigemina 
and T. buffelt). Then A. marginale real-time PCR was modified by adding a primer/probe 
set specific for A. centrale, thus obtaining a duplex assay for simultaneous detection of 
both Anaplasma spp. in the same reaction. Duplex real-time PCR has proven to be a 
powerful tool for differentiating between closely related infectious agents (Decaro et al. , 
2006a; 2006b) and between vaccine and field strains of the same genotype (Decaro et 
al., 2006c; Elia et al., 2008). 
1.10 Treatment, prevention and control strategies 
Control measures for anaplasmosis vary with different geographic locations, including 
maintenance of anaplasmosis free herds, control of tick vectors, administration of 
antibiotics and vaccination (de Waal, 2000). Dairy and beef cattle farmers have relied 
on dipping measures for control of tick infestation; however, in areas where tick vectors 
are well-established, the continuous exposure to ticks leads to endemic stability 
situation (de Waal, 2000). Vector control is labour intensive, expensive and 
environmental pollution is also a major concern. 
19 
Prophylaxis has been through administration of antibiotics. Chemotherapy is intended 
for prevention of clinical anaplasmosis but it does not prevent cattle from becoming 
persistently infected with A. marginale; however, cattle receiving antibiotics therapy may 
not be cleared of infection. Tetracycline administration is accompanied by 
disadvantages of expenses, and the demand of continuous feed ing and also the risk of 
development of resistant Anaplasma organisms, although the resistance of A. marginale 
to antibiotics has not been reported (de Waal, 2000). Kocan et al. , (2003) reviewed the 
progression and history of vaccine development which included live and killed vaccines. 
Both vaccines have relied on A. marginale infected bovine erythrocytes as source of 
antigen. 
The live and killed vaccines induce protective immunity that diminishes the cl inical 
signs, but does not prevent cattle from becoming persistently infected with A. marginale. 
Live vaccine (A centrale), which was introduced by Sir Arnold Theiler has been used 
widely in South Africa but the vaccination renders partial protection against A. marginale 
and its success vary according to genotypes of A. marginale (Brown et al. , 1998; de 
Waal, 2000). In contrast, trials conducted in South America and Africa with heterologous 
strains showed low efficacy of this vaccine (Turton et al., 1998; Bock and de Vos, 2001 ; 
Dark et al. , 2011 ). 
Major surface proteins (MSP) have been manipulatively used experimentally as vaccine 
candidates against A. marginale infections (Palmer et al., 1987; Allred et al. , 1990). 
MSP1 a has an ability to induce T-cell response and contains conserved B-cell epitope 
in the repeated peptides that is recognized by immunized and protected cattle (Kocan et 
al. , 2003, de la Fuente et al. , 2003b ). Experiments with recombinant MSP1 a have 
shown partial protection against anaplasmosis in cattle (Torina et al., 2014). 
20 
Recent studies have identified members of the pfam01617 from msp2 family to be 
possible vaccine candidates because most of them are found in cross-linked surface 
antigen complexes with the other members of pfam01617 encoding conserved outer 
membrane proteins, which are expressed in A. marginale (Noh et al. , 2006; Dark et al., 
2011 ). 
1.11 Anap/asma marginale major surface proteins and their role in host-vector-
pathogen interactions 
The outer membrane proteins of A. marginale have been the focus of direct research to 
obtain an improved vaccine against bovine anaplasmosis (Palmer et al. , 1999). 
Immunization with purified outer membranes induces protection against acute A. 
marginale infection and disease (Tebele et al. , 1991 ). Various major outer membranes 
have been described based on the proteomic and genomic approach , and 21 proteins 
were identified within the outer membrane immunogen (Lopez et al. , 2005). Some well-
characterized outer membranes proteins designated major surface proteins (MSPs): 
msp1a, msp1b, msp2, msp3, msp4 and msp5 were evaluated as potential candidates 
for antigens of vaccine production and diagnostic evaluations (Visser et al. , 1992; 
Oberle et al. , 1993; McGarey et al., 1994; Alleman and Barbet, 1996; Kano et al., 2002; 
Garcia-Garcia et al. , 2004a). 
21 
1.11.1 The MSP1 Complex 
The relationship of the rickettsia with the bovine host is complex as it has interaction 
with the erythrocyte and endothelial cells (Carreno et al., 2007). A number of rickettsial 
protein and the bovine immune system have not been studied in detai l yet, there is no 
final solution (vaccine or otherwise). Several major surface proteins (MSPs) have been 
identified in Anaplasma spp., which have been most extensively characterized in A. 
marginale (Palmer et al. , 1985; de la Fuente et al. , 2001 a, 2005b; Kocan et al. , 2003; 
2004; 2008). Anaplasma MSPs are involved in interactions with both vertebrates and 
invertebrates host cells (de la Fuente et al. , 2001a; 2005a; Kocan et al., 2003; 2004; 
Brayton et al. , 2005; Dunning Hotopp et al., 2006; Nelson et al., 2008), and are likely to 
evolve more rapidly than other genes because they are subjected to selective pressures 
exerted by host immune systems. 
Six MPSs have been identified in A. marginale from cattle and ticks of which three, 
msp1a, msp4 and msp5, are coded by single gene and do not vary within isolates 
(Bowie et al. , 2002). The other three, msp1b, msp2 and msp3, are from multigene 
families and may vary antigenetically in persistently infected cattle (Barbet et al. , 2000; 
Kocan et al. , 2000; Bowie et al., 2002). Major surface protein 1 is a heterodimer 
composed of two structurally unrelated polypeptides: msp1a, (100kDa), which is 
encoded by a single gene, msp1a (Lew et al. , 2002) and msp1b (105 kDa), which is 
encoded by at least two genes, msp1f31 and msp1f32 (Barbet et al., 1987; Viseshakul et 
al. , 2000; Camacho-Nuez et al., 2000; Bowie et al., 2002; Macmillan et al. , 2006). The 
msp1a, and msp1b proteins form non-covalent dimers and are exposed on the surface 
of A. marginale (Barbet et al., 1987). However, only a single msp1b protein, msp1b1 , 
was identified within the MSP1 complex (Macmillan et al. , 2006). 
22 
The molecular weight of msp1a varies in size among isolates due to different numbers 
of tandemly repeated 23-31 amino acid peptides and has been used for identification of 
geographic strains (Allred et al. , 1990; de la Fuente et al. , 2001 a; 2003a; 2005a; 
2007a). The msp1a, tandem repeats are located after a conserved decapeptide in the 
amino terminal region of the protein and are exposed extracellularly for interaction with 
host cell receptors (de la Fuente et al., 2003a). The frequency of variable amino acid 
positions within geographic isolates is higher in this region than in the rest of the protein 
(de la Fuente et al. , 2001 a). 
Functionally, msp1a was shown to be an adhesin for bovine erythrocytes and tick cells 
and the adhesin domain was identified on the variable N-terminal region contain ing the 
repeated peptides (McGarey and Allred, 1994; McGarey et al. , 1994; de la Fuente et al., 
2001 b). It was also shown to be involved in the transmission of A. marginale by 
Dermacentor spp. (de la Fuente et al. , 2001 c) and to be differentially regulated in tick 
cells and bovine erythrocytes (Garcia-Garcia et al., 2004b). The msp1a, although 
variable in the number of repeated peptides, induces strong T-cell responses and 
contains a conserved B-cell epitope in the repeated peptides that is recognized by 
immunized and protected cattle (Kocan et al., 2003; de la Fuente et al., 2005a). The 
msp1a also contains Th1 cell epitopes in the conserved region, which may be involved 
in immunoprotection (Brown et al., 2001). 
The msp1b, encoded by at least two genes, msp1b and msp1b2, is polymorphic among 
geographic isolates of A. marginale (Barbet et al. , 1987; Camacho Nuez et al. , 2000; 
Viseshakul et al. , 2000; Bowie et al., 2002). Although msp1b is encoded by a multigene 
family, only small variations in protein sequences of msp1b and msp1b2 were observed 
during the life cycle of the Rickettsia in cattle and ticks (Bowie et al. , 2002). This protein, 
which forms a complex with msp1a, is an adhesin for bovine erythrocytes (McGarey and 
Allred , 1994; McGarey et al., 1994). However, MSP1b was recently demonstrated to be 
adhesin only for bovine erythrocytes and did not prove to be an adhesin for tick cells (de 
la Fuente et al. , 2001 a). 
23 
The A. marginale surface is characterized by the presence of two highly abundant and 
closely related outer membrane proteins Major Surface Protein 2 (msp2) and (msp3) 
(Brayton et al., 2001 ). The predominant immune responses are generated against these 
two proteins (McGuire et al. , 1991 ; Brown et al. , 2001 ; 2003). However, both msp2 and 
msp3 are highly antigenically variable, both within an infection and between strains 
(McGuire et al., 1984; French et al., 1998; 1999; Rodriguez et al., 2005). 
Thus, while antibody response to msp2 and msp3 antigenic variants plays a key role in 
how persistent infection is established and the population strain structure, these 
abundant surface proteins are not targets for development of a widely cross-protective 
vaccine and anti- msp2/msp3 immune responses do not associate with protective 
efficacy of the outer membrane vaccine (Palmer et al. , 2009; Noh et al. , 2010). Using 
genomic and proteomic approaches, the minor components of the outer membrane 
protein immunogen have been identified (Noh et al., 2006; 2008; Brayton et al. , 2005; 
Lopez et al. , 2005; 2008; Agnes et al. , 2011 ). 
Although markedly less abundant, these minor proteins are invariant during infection 
and highly conserved among strains-thus representing much more attractive targets fo r 
vaccine development. Importantly, the proteomic identification within the outer 
membrane immunogen and the bioinformatics prediction of surface localization was 
confirmed for a subset of these proteins by surface-specific cross-linking (Noh et al. , 
2008). The isolated cross-linked surface protein complex induced protection equal to 
that of the native outer membrane immunogen (Noh et al., 2008). 
24 
The msp2 and msp3 are both encoded by large polymorphic, multigene families 
(Palmer et al. , 1994; Alleman et al., 1997). The msp2 sequence and antigenic 
composition varies during cyclic rickettsemia in cattle (French et al. , 1998; 1999; Barbet 
et al., 2001) and in persistently infected ticks (de la Fuente et al. , 2001 a). MSP2 is 
encoded on a polycistronic mRNA. The msp2 gene within the expression site is 
polymorphic. The msp2 encodes numerous amino acid sequence variants selected in 
bovine erythrocytic and tick salivary gland populations of A. marginale (French et al. , 
1998; 1999; Barbet et al., 2000; Brayton et al., 2001 ; de la Fuente et al. , 2001 a; Meeus 
and Barbet, 2001 ). 
The msp3 also varies in antigenic properties and structure among geographic isolates 
(Alleman and Barbet, 1996). The msp2 and msp3 are involved in the induction of a 
protective bovine immune response to A. marginale (Palmer et al. , 1999). 
The msp4 and msp5 are encoded by single copy genes. Although msp4 is highly 
conserved (Oberle and Barbet, 1993; de al Fuente et al. , 2003a), information about its 
function is not available. 
The msp5 is highly conserved surface protein that has been proven effective as a 
diagnostic antigen and used in a competitive enzyme linked immunosorbent assay 
(cELISA) commercially available in the United States (Torioni De Echaide et al. , 1998). 
The function of msp5 is also unknown. The msp2 operon-associated genes OpAG1 , 
OpAG2, and OpAG3, have been identified in A. marginale and may encode for surface 
proteins (Lohr et al. , 2002). 
25 
1.11.2 Vector-pathogen relationships 
1.11.2.1 Anaplasma MSPs and vector -pathogen interactions 
The evolutionary history of vector-pathogen interactions could be reflected in the 
sequence variation of the Anaplasma major surface proteins. Previous studies 
demonstrated that A. marginale msp1a, but not msp4 is under positive selection 
pressure (de la Fuente et al., 2003a). Initial analysis of A. marginale msp1a and 
Dermacentor variabilis 16S rDNA sequences from various areas of the United States 
suggested tick-pathogen co-evolution (de la Fuente et al., 2001 d) consistent with the 
biological function of MSP1a involved in the transmission of A. marginale by ticks (de la 
Fuente et al. , 2001 c). However, the genetic diversity of sequences complicates the 
study of tick-pathogen co-evolution (de la Fuente et al. , 2010). 
Anaplasma marginale strains are apparently not transmissible by ticks and rely on 
mechanical transmission for completion of the life cycle in nature (de la Fuente et al., 
2005d; Hornok et al., 2008). These facts pose the question of what evolutionary 
adaptations may have occurred to insure an efficient mechanical transmission of non-
tick-transmissible A. marginale strains. Recently, Scoles et al. , (2005) concluded that 
biological transmission by ticks is more efficient than mechanical transmission of A. 
marginale (de la Fuente et al. , 2010). 
The adhesin domain of A. marginale msp1a has been identified on the extracellular N-
terminal region of the protein that conta ins the repeated peptides (de la Fuente et al., 
2003b). The binding of msp1a to tick cell extract (TCE) was observed within the msp1a 
tandem repeats, the negatively charged amino acids, aspartic acid (0) and glutamic 
acid (E), at position 20 demonstrated that peptide containing acidic amino acids D or E 
at position 20 bound to TCE, while peptides with G as the 201h amino acid were not 
adhesive to TCE (de la Fuente et al., 2003b). 
26 
1.12 Phylogenetic relationships of geographic isolates of A. marginale 
A phylogenetic tree is an estimate of the relationships among taxa (or sequences) and 
their hypothetical common ancestors (Nei and Kumar 2000; Felsenstein 2004; Hall 
2011 ). Today most phylogenetic trees are built from molecular data: DNA or protein 
sequences. Originally, the purpose of most molecular phylogenetic trees was to 
estimate the relationships among the species represented by those sequences, but 
today the purposes have expanded to include understanding the relationships among 
the sequences themselves without regard to the host species, inferring the functions of 
genes that have not been studied experimentally (Hall et al., 2009), and elucidating 
mechanisms that lead to microbial outbreaks (Hall and Barlow 2006) among many 
others. Building a phylogenetic tree requires four distinct steps : (Step 1) identify and 
acquire a set of homologous DNA or protein sequences, (Step 2) align those 
sequences, (Step 3) estimate a tree from the aligned sequences, and (Step 4) present 
that tree in such a way as to clearly convey the relevant information to others (Hall, 
2013). 
Phylogenetic analysis of A. marginale geographic isolates from the United States was 
performed using the genes msp1a and msp4 (de la Fuente et al., 2001d). The results of 
these analyses strongly support a southeastern clade of A. marginale composed of 
isolates from Virginia and Florida. Analysis of 16S rDNA fragment sequences from the 
tick vector of A. marginale, D. variabilis, from various areas of the United States was 
performed and suggested coevolution of the vector and pathogen (de la Fuente et al., 
2001d). 
Isolates of A. marginale from the United States also grouped into two clades, southern 
clade consisting of isolates from Florida, Mississippi , and Virgin ia, and a west-central 
clade consisting of isolates from California, Idaho, Illinois, Oklahoma, and Texas. 
Although little phylogeographic resolution was detected within any of these higher 
clades, msp4 sequences appear to be a good genetic marker for inferring 
phylogeographic patterns of isolates of A. marginale on a broad geographic scale . 
27 
In contrast to the phylogeographic resolution provided by msp4, DNA and protein 
sequence variation from msp1a representing 20 New World isolates of A. marginale 
failed to provide phylogeographic resolution (de la Fuente et al. , 2007b). Most variation 
in msp1a sequences appeared unique to a given isolate. In fact, simi lar DNA sequence 
variation in msp1a was detected within isolates from Idaho and Florida and from Idaho 
and Argentina. These results suggest that the msp 1a  sequence may be rapidly evolving 
and that the msp1a gene may provide phylogeographic information only when 
numerous msp1a sequences from a given area are included in the analysis (de la 
Fuente et al. , 2007a). 
The analysis of msp 1a  DNA and protein sequences demonstrated extensive genotypic 
variation among Oklahoma isolates of A. marginale and failed to provide 
phylogeographic resolution within Oklahoma or on a broader scale, including isolates 
from other United States and Latin America. Furthermore, analysis of codon and amino 
acid changes over the msp1a and msp4 phylogenies provided evidence that msp1a but 
not msp4, is under positive selection pressure. These results suggest that even if 
msp1a sequences are rapidly evolving, MSP1a genotypes reflect the history of cattle 
movement more than the geographic distribution of A. marginale isolates. Research 
analysis suggest that different A. marginale genotypes are maintained within a herd in 
an area of endemic infection by independent transmission events and that infection with 
more than one genotype per host is prevented, a phenomenon described as infection 
exclusion (de la Fuente et al., 2003a). 
Therefore, if cattle movements import a new A. marginale genotype, it could be 
established by mechanical and/ or biological transmission to susceptible cattle. In 
regions with few cattle introductions, like Australia, little genotypic variation are found 
within A. marginale isolates (de la Fuente et al. , 2005a). 
28 
CHAPTER 2 
OBJECTIVES OF THE STUDY 
2.1 Statement of the problem 
Anaplasma marginale genotypes have been well characterized and are highly variable 
in endemic areas worldwide (de la Fuente et al., 2010) . Anaplasma marginale is quite 
host specific for ruminants and anaplasmosis occurs primarily in cattle; other ruminants 
may serve as reservoirs of infection. While A. marginale is transm itted biologically by 
ticks, mechanical transmission by blood-contaminated mouthparts of biting flies or 
fomites also frequently occurs. Mechanical transmission may be the only means of 
spreading anaplasmosis in areas where tick vectors are absent or are unable to 
transmit the local A. marginale strain (Kocan et al., 2010). 
Immunization of cattle with affinity- purified native MSP1 complex induced protective 
immunity in cattle that received homologous or heterologous challenge with A. 
marginale geographic isolates (Palmer et al., 1987; 1989). The msp1a has been shown 
to have a neutralization sensitive epitope (Palmer et al., 1987) and to be an A. 
marginale adhesin for both bovine erythrocytes and tick cells, while msp1b1 is an 
adhesin only for bovine erythrocytes (McGarey et al., 1994; McGarey and Allred , 1994; 
de la Fuente et al. , 2010). Killed vaccine marketed previously in the USA used A. 
marginale antigen that was partially purified from bovine erythrocytes (Brock et al. , 
1965; Hart et al., 1981 ; Mccorkle-Shirley et al., 1985). This vaccine was used effectively 
until it was removed from the market in 1999 (Kocan et al., 2010). 
These blood-derived killed vaccines reduced clinical anaplasmosis and were expensive 
to produce, difficult to standardize and often not cross-protective in widely separated 
geographic areas with different endemic A. marginale isolates. The blood-derived 
vaccines also bore risk of being contaminated with bovine cells or pathogens that 
frequently cause persistent infections in cattle (Palmer, 1989; Kocan et al., 2000). 
29 
Despite the importance of anaplasmosis in South Africa and other African countries, A. 
marginale strains have not been genetically characterized in Africa (Mtshali et al. , 
2007). Vaccination experiments with recombinant msp1a have resulted in partial 
protection against clinical anaplasmosis in cattle and reduced infection levels in ticks, 
thus supporting the inclusion of msp1 a in vaccines for the control of bovine 
anaplasmosis (Kocan et al., 2010). 
The phylogenetic analysis of A. marginale strains using MSPs has been recently 
reviewed. These analyses suggest that MSPs may not be good markers for 
biogeographically studies on a global scale. However, they may be useful for strain 
comparison in given regions and could provide information about the evolution of host-
pathogen and vector-pathogen relationships (Cabezas-Cruz et al., 2013). 
While a universal vaccine has yet to be developed for anaplasmosis, preliminary 
analysis of the phylogeographic relationships of A. marginale based on two major 
surface proteins (msp1a and msp4) has shown phylogeographic partitioning of parasite 
isolates in the United States (de la Fuente et al. , 2007a). This information supports the 
inclusion of several geographic isolates of A. marginale in development of vaccine 
formulations for the United States (Kocan et al., 2010). Recent research has focused on 
MSPs that may be used to elucidate phylogeographic patterns of A. marginale (de la 
Fuente et al. , 2001 a) and that are involved in interactions with vertebrate and 
invertebrate host cells (McGarey et al., 1994; McGarey and Allred, 1994; de la Fuente 
et al. , 2001a). 
30 
These MSPs involved in host-pathogen interactions may evolve more rapidly than other 
nuclear genes because of selective pressures exerted by host immune systems (de la 
Fuente et al., 2010). Of the six A. marginale MSPs that have been identified and 
characterized , only three (msp1a, msp4 and msp5) are each encoded by a single gene. 
Because these MSPs do not appear to undergo antigenic variation in cattle or ticks, 
they were reported to be more stable genes for phylogenetic studies (Bowie et al. , 
2002). 
Of these three MSPs, msp1a has been reported to be an adhesin for bovine 
erythrocytes and tick cells and to effect infection and transmission of A. marginale by 
Dermacentor spp. ticks (McGarey et al. , 1994; McGarey and Allred , 1994; de la Fuente 
et al., 2001 b, 2001 c). Although the specific function of msp4 is currently is not known , 
the previous analysis of the gene from A. marginale isolates detected sufficient 
sequence variation to support its use in phylogeographic studies (de la Fuente et al., 
2001 a). Therefore, msp4 warranted further testing for use in detection of 
phylogeographic patterns among A. marginale isolates. 
In a previous report of the phylogeographic relationships of A. marginale isolates from 
the United States (de la Fuente et al., 2001a). It was found that DNA sequence variation 
in msp4 provided phylogenetic resolution for intraspecific relationships among isolates 
(de la Fuente et al. , 2002b). In addition, when DNA sequence variation of msp1a was 
interpreted in combination with msp4 phylogeny, important phylogeographic information 
became apparent. This study utilised MSPs to explore the epidemiology and phylogeny 
of A. marginale causing anaplasmosis in selected South African provinces. 
31 
2.2Aim of the study 
The aim of this study is to determine the prevalence, genetic distribution and 
phylogenetic relationship of A. marginale infecting South African-cattle by using msp1a 
and msp4 gene 
2.3 Objectives 
2.3.1 To determine the prevalence of A. marginale infecting cattle in South Africa by 
using msp1a as a target gene. 
2.3.2 To establish genetic diversity of A. marginale infecting cattle in South Africa using 
msp1a as marker gene. 
2.3.3 To determine phylogenetic relationship of A. marginale infecting South African 
cattle by using msp1a and msp4 genes. 
32 
CHAPTER 3 
MOLECULAR DETECTION OF ANAPLASMA MARG/NALE INFECTING 
CATTLE IN SOUTH AFRICA BY PCR TARGETING msp1a GENE 
3.1 Introduction 
Bovine anaplasmosis is a tick-borne hemoparasitic disease caused by Anaplasma 
marginale and is the most widely distributed disease of cattle in South Africa (de Waal, 
2000; Ndou et al., 2010). Economic losses associated with anaplasmosis in cattle 
include impaired production , mortality and expenses in control measures (Regassa et 
al., 2003). 
Ticks and the diseases they transmit have been identified as the major cause of 
widespread morbidity and mortality in cattle kept by smallfarm holders in the semiarid 
areas of South Africa (Dold and Cocks, 2001 ; Mapiye et al. , 2009). Poor cattle health 
management, resistance of ticks to most acaricides and the use of inappropriate cattle 
breeds (Dold and Cocks, 2001 ; Marufu et al. , 2011 ) have increased the prevalence of 
ticks and tick-borne diseases (TBD) in smallholder cattle herds. 
Polymerase chain reaction detection methods have been developed. These 
tests/assays are extremely sensitive and specific in the detection of A. marginale 
infections in cattle (Lew et al., 2002; de la Fuente et al., 2005b; Malad et al. , 2006). 
Some of these assays, such as the real-time PCR, were developed to enable 
simultaneous detection and quantification of the A. marginale DNA in bovine blood. This 
is essential in supporting the clinical diagnosis, assessing the carrier status of the cattle 
and evaluating the efficacy of vaccines and antirickettsial drugs (Carelli et al., 2007). 
33 
Among the genes encoding for major surface proteins in A. marginale, msp1a has been 
extensively used for strain characterization (Cabezas-Cruz et al. , 2013). The protein 
msp 1a  is involved in the interaction of the bacterium with vertebrate and invertebrate 
host cells (de la Fuente et al. , 2010). Several strains of A. marginale have been 
identified worldwide and these strains differ in their morphology, msp1a amino acid 
sequence, antigenic characteristics, and ability to be transmitted by ticks (de la Fuente 
et al., 2007a; Estrada-Pena et al., 2009). 
The msp 1a  gene is thus a good candidate for the development of a generic A. 
marginale genotyping assay that could be applied globally and also to track strains (Lew 
et al. , 2002). The objective of the study was to determine the prevalence of A. marginale 
infecting cattle from Limpopo, Mpumalanga, North West, Gauteng, KwaZulu-Natal, 
Eastern Cape, Western Cape and Northern Cape provinces of South Africa by PCR 
using the msp1a gene. 
3.2 Materials and methods 
3.2.1. Study site and sample collection 
Two hundred and eighty samples were collected from cattle in several South African 
provinces and the exact locations with coordinates are provided in Figure 5 and Table 4. 
Farms with different productive systems were chosen. Cattle were selected on a 
random basis following their owner's approval for the collection of blood samples. Blood 
samples were obtained from cattle and no stratification according to age or sex was 
applied. No vaccination histories were available for these animals. Tail venupuncture 
was performed using an 18 gauge needle and blood samples were collected into sterile 
vacutainer tubes (10 ml) with EDTA and were kept at 4 °C until delivered at the 
laboratory. 
34 
3.2.2 DNA extraction 
The genomic DNA was extracted from cattle blood samples using ZR Genomic DNA rM 
Tissue Miniprep (Zymo Research, CA, USA). Total volume of blood sample was 
adjusted in a microcentrifuge tube with water to 100 µI prior to adding 95 µI of 2x 
Digestion buffer and 5 µI of Proteinase K. The mixture was thoroughly mixed and then 
incubated at 55 °C for 20 minutes. A volume of 700 µI of genomic lysis buffer was 
added to the tube and mixed thoroughly by vortexing. 
The mixture was carefully transferred to a Zymo-Spin™ llC column in a collection tube 
and centrifuged at 10,000 x g for one minute. A volume of 200 µI of DNA pre-wash 
buffer was added to the spin column in a new collection tube and centrifuged at 10,000 
x g for one minute. A volume of 400 µI of g-DNA wash buffer was added to the spin 
column and centrifuged at 10, 000 x g for one minute. The spin columns were carefully 
transferred to a clean 1.5 ml microcentrifuge tube and 100 µI of DNA Elution buffer 
added to the spin column. The microcentrifuge tube was incubated for 5 minutes at 
room temperature, and then centrifuged at 8000 rpm for 30 seconds to elute the DNA. 
The eluted DNA was stored at -20 °C until used for studies. The concentration of DNA 
was measured using a NanoDrop® ND-1000 (NanoDrop Technologies Inc., Wi lmington, 
USA). 
3.2.3. Polymerase chain reaction (PCR) 
A specific set of primers was used to amplify msp 1a  gene. A PCR was performed using 
the primers that were previously designed to amplify a fragment of msp1a gene, 
contain ing the tandem repeat region. Primers were synthesized and supplied by lnqaba 
Biotech (lnqaba Biotechnical Industries (Pty) Ltd, Pretoria, Gauteng, South Africa). The 
PCR reagents mixture consisted of total volume of 25 µI of Thermo Scientific DreamTaq 
Green PCR Master Mix (2X), 10 µM of forward and reverse primers, 1 µg Template 
DNA in 0.2 ml PCR tubes. 
35 
The PCR mixture was then amplified by DNA thermocycler (Bio-Rad T100™ DNA 
Thermal Cycler, Bio-Rad Laboratories, Johannesburg, South Africa). The amplification 
process involved an initial denaturation at 94 °C for 3 minutes followed by 40 cycles of 
denaturation at 94 °C for 1 minute at 94 °C, annealing at 65 °C for 1 minute , and an 
extension at 72 °C for 2 minutes. A cycle of a final extension was done at 72 °C for 7 
minutes. The amplified products were then separated by electrophoresis using 1 % 
agarose gel immersed in TSE buffer (89 mM Tris-Borate, 2 mM EDTA, pH 8). One kilo-
base pair ladder marker was used to determine the size of the PCR products after 
staining with 0.5 µg/ml GelRed (Life Sciences, Fermentas GmbH, Germany) and 
visualization under UV illumination. Gel documentation was done and taking a 
photograph using a camera (Figure 3). 
Table 3: Oligonucleotide sequences of primers used in this study 
Primer designation Oligonucleotide (5'-3') References 
1733F TGTGCTTATGGCAGACATTTCC Lew et al. , 2002 
2957R AAACCTTGTAGCCCCAACTTATCC Lew et al., 2002 
3.2.4. Statistical analysis 
Statistical analysis of the results were performed at the Department of Biostatistics and 
Statistics at the Medical Research Council , Pretoria Unit using the Fisher's exact and 
Chi-square test to test whether the prevalence is associated with the type of farm while 
chi-squared test was used to evaluate any significant difference (P=0.0001 ). The 
confidence intervals and standard errors of the prevalence of Anaplasma marginale 
infections in cattle were calculated at 95% using Kruskal-Wallis equality of population 
rank test. Pearson chi-square test was used to analyse PCR results based on 
frequency, row, column and cell percentage (Appendix 1) . 
36 
3.3. Results 
3.3.1 Detection of A. marginale infections 
Two hundred and eighty blood samples were analysed by PCR technique. The primers 
1733F and 2957R were used to amplify a fragment of msp 1a  gene, contain ing the 
tandem repeat region which varies among isolates. In this study PCR amplicons of 
variable sizes were obtained, ranging from 630 to 1100 bp corresponding to msp1a 
gene of different molecular weight as shown previously by Lew (2002). Two hundred 
and nine cattle blood samples out of 280 were PCR positive for A. marginale infection 
as shown in Appendix 1. 
3.3.2 Molecular prevalence of A. margina/e isolates infecting cattle in South 
African provinces 
The PCR results of A. marginale infection ranged from 0% to 100% for samples from 
Mpumalanga and Gauteng. Eastern Cape comprised of 100%, KwaZulu-Natal 87.27%, 
Western Cape 84.62%, Limpopo 80% and North West 78. 79% positive infectious rates. 
None of the Northern Cape samples were positive by PCR (Appendix 1 ). 
3.3.3 Statistical prevalence of A. marginale isolates infecting cattle in South 
African provinces 
Statistical analysis was conducted to obtain the prevalence rates using the PCR results. 
The highest relative prevalence was observed in KwaZulu-Natal with 23.0%, second 
Eastern Cape 19.1% , then Limpopo 15.3%, North West 12.4%, Western Cape 10.5%, 
Mpumalanga 10.1%  and Gauteng 9.6%. These were obtained by considering relative 
rates of prevalence with in the province. There was no significant statistical association 
between prevalence of A. marginale with the type of farm (communal or commercial). 
37 
PCR products with single bands as shown in Figure 3 were sequenced. The single PCR 
products from the seven different geographical regions including Limpopo, 
Mpumalanga, North West, Gauteng, KwaZulu-Natal, Eastern Cape and Western Cape 
of South African provinces A. marginale were sequenced and forty four gene sequences 
were submitted to GenBank (Appendix 2). BLAST analysis confirmed that the DNA 
results were indeed A. marginale msp1a gene and showed high identity (90-100%) with 
A. marginale msp1a deposited in GenBank. 
38 
Although A. marginale was detected in some of the samples as indicated by PCR 
results, most of the positive PCR samples from Mpumalanga, KwaZulu-Natal and 
Eastern Cape provinces, had non-specific bands and were not further analysed (Figure 
3). 
M 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 
10000 
5000 
3000 
2000 
1200 
850 
500 
300 
100 
Figure 3: Gel image of 1 % agarose gel electrophoresis of PCR amplification products obtained 
from A. marginale isolates from Western Cape Province in South Africa using 1733F and 2973R 
primers (Lew et al., 2002). Lane Marker (M): GeneRuler™ I Kb DNA ladder, ready-to-use; Lane 
1-5, 7-8, 13-14, 16-18 indicate positive results and lane 6, 9-12, 15 indicate negative results; lane 
19 negative control. 
39 
60.0 
50.0 
40.0 
VJ 
2 
Ill 30.0 
0 23.0 
.!!1 19.1 
20.0 
.91 15.3 12.4 
.Isl:l  9.6 10.1 10.5 
e> 10.0 
Ill 
E 0.0 
~ 0.0 
L I I I I I I 
0 e ~~ ~ 
c.l~ ~ ~o ~e; ~~~  .:f.e<} 
~e ~e 
Q) ~ ~o <.J <.J 0 I..~ ~~ c ~ 
(I) ._e; <> ~~ ~~ ,l' ~~ I..~ ._(:' ~e; 
(ij ~'b. . ~ ~o .:t-e" 
> ~.:J. 
~~ ~o~ 
~ 
Cl. 
• Positiw 
Figure 4: Prevalence of A. marginale by province in South Africa. 
40 
3.4 Discussion 
It has been reported that bovine anaplasmosis is one of the most important cause of 
cattle mortalities in low-input farming areas in South Africa (Mapiye et al. , 2009). 
Polymerase chain reaction detection methods have been developed, which are 
extremely sensitive and specific for the detection of A. marginale infections in cattle (de 
la Fuente et al., 2005b; Molad et al. , 2006). It is crucial to use sensitive and efficient 
molecular techniques to generate accurate information on the prevalence of A. 
marginale, which are crucial not only for developing appropriate control measures but 
for providing an understanding of host resistance in different cattle genotypes (Marufu, 
2014). 
In this study, a sensitive and specific msp1a PCR (Lew et al. , 2002) was used to 
determine the presence of A. marginale in eight different provinces of South Africa 
(Appendix 1) . The results indicate the presence of A. marginale infection in these 
provinces with an overall prevalence of 76% except for Northern Cape in which the 
prevalence was 0%. This prevalence may be considered as high as in other endemic 
regions of the world. 
Recently, 70% prevalence of A. marginale in a cattle herd was reported in Brazil (Pohl 
et al. , 2013). Another study by de la Fuente et al., (2005a) showed a prevalence range 
from 25 to 100% for A. marginale infecting cattle herds in Italy. Results of this study 
confirm that bovine anaplasmosis is endemic and highly prevalent in South Africa . 
The absence of positive samples in Northern Cape Province may be explained by 
special climate conditions in th is area that limits the survival of tick vectors (Mtshali and 
Mtshali , 2013). However, the prevalence of bovine anaplasmosis at certa in areas is a 
complex process also involving bovine population immunity to A. marginale, tick control 
practices, tick transmission, grazing system as well as the genetic variability of A. 
marginale at the population level (Hamou et al., 2012). 
41 
Despite high prevalence of A. marginale in the studied area, the veterinary authority 
(State Veterinary officials) has not reported clinical disease in cattle, which is an 
indication of endemic stabil ity . Endemic stability is an epidemiological state, in which 
clinical disease is scarce despite high levels of infection in the population (Coleman et 
al. , 2001 ; Jonsson et al., 2012). 
This is the first report of A. marginale prevalence at the national level based on 
molecular data. The results confirm endemicity and high prevalence of A. marginale in 
South Africa. Further investigations should address the influence of such high 
prevalence in cattle in South Africa . This will help in the understanding of the 
epidemiology and the use of appropriate control and prevention measures to reduce the 
high prevalence in cattle in South Africa before experiencing an outbreak. 
42 
CHAPTER4 
EPIDEMIOLOGY AND EVOLUTION OF THE GENETIC VARIABILITY OF 
ANAPLASMA MARG/NALE IN SOUTH AFRICA 
This work has been published as follows: 
A.M. Mutshembele, A. Cabezas-Cruz, M.S. Mtshali, O.M.M. Thekisoe, R.C. Galindo 
and J. de la Fuente. 2014. Epidemiology and evolution of the genetic variabi lity of 
Anaplasma marginale in South Africa. Ticks and Tick -borne diseases, 5 (6): 624-631 . 
4.1 Introduction 
Bovine anaplasmosis is a non-contagious tick-borne disease caused by infection of 
cattle with A. marginale, an obligate intraerythrocytic bacterium classified in the family 
Anaplasmataceae, order Rickettsiales (Dumler et al. , 2001 ). This pathogen is 
transmitted biologically by ticks, mechanically by biting insects and blood-contaminated 
fomites and from cow to calf via transplacental transmission (Aubry and Geale, 2011 ). 
Five tick species have been shown experimentally to transmit A. marginale in South 
Africa, including Rhipicephalus microplus, R. decoloratus, R. evertsi evertsi, R. simus 
and Hyalomma marginatum rufipes (as reviewed by de Waal. , 2000). 
Acute disease in cattle is characterized by weight loss, fever, abortion, low milk 
production and in some cases death; the animals that recover from the disease become 
persistently infected and serve as reservoir of infection for mechanical transmission and 
biological transmission by ticks (Kocan et al., 2003). Anaplasmosis is widespread in 
South Africa and, as estimated by de Waal. (2000), 99% of the total cattle populat ion is 
at risk of acquiring A. marginale infection . 
43 
Currently, antimicrobial drugs are not available for the elimination of persistent 
infections in cattle. Although the World Organization for Animal Health proposed the use 
of enrofloxacin, imidocarb, and oxytetracycline for the elimination of persistent A. 
marginale infections in cattle, these antimicrobial drugs were not found to eliminate the 
persistent A. marginale infections (Coetzee et al., 2005; 2006). 
Vaccines have been used as an alternative method for control of anaplasmosis. A live 
vaccine using A. marginale sub sp centrale (A. centrale), a subspecies of relatively low 
pathogenicity, is available in South Africa, Australia, Israel and Latin America but this 
vaccine has proven to be only partially effective and reports of vaccination failure are 
not uncommon (Kocan et al. , 2003). While anaplasmosis still constitutes a problem for 
cattle production in South Africa, sales of vaccines for bovine anaplasmosis in South 
Africa have reduced from 800, 000 to 200, 000 doses over 22 years (1976-1998) (de 
Waal. , 2000) . 
MSP1 a has been shown to have potential use as a vaccine antigen because this protein 
contains both neutralization sensitive (Palmer et al. , 1987; Allred et al., 1990) and 
immunodominant epitopes (Garcia-Garcia et al., 2004b). Recently, use of MSP1 a for 
vaccine development for A. marginale has regained new attention. The N-terminus 
tandem repeated reg ion of this protein was used in immunization trials in cattle against 
A. marginale (Torina et al., 2014) and laboratory animal models (Santos et al., 2013; 
Silvestre et al., 2014 ), and demonstrated promising results . MSP1 a is one of six MSPs 
that have been described in A. marginale. This protein is encoded by a single-copy 
gene, msp1a, which is conserved during the multiplication of the parasite in cattle and 
ticks (Kocan et al. , 2003) and has been useful for epidemiological studies of A. 
marginale in various regions of the world (Ruybal et al. , 2009; Almazan et al., 2008). 
44 
r--------------------------------------------------
MSP1 a contains tandem repeats at the N-terminus of the protein which present 
functional residues that serve as adhesins for bovine erythrocytes and tick cells, a 
prerequisite for infection of host cells (McGarey et al. , 1994; de la Fuente et al., 2003a). 
While the tandem repeats of MSP1a are highly variable, repeats are commonly 
represented among worldwide strains (Cabezas-Cruz et al., 201 3) and have been 
shown to evolve under positive selection (de la Fuente et al., 2003b). 
However, the specific codon positions that evolve under positive or negative selection 
have not been reported. For development of MSP1a-based vaccines, some 
characteristics of MSP1 a should be taken into consideration , including (i) the extant 
genetic variability of MSP1 a, (ii) the evolution of MSP1 a genetic diversity, and (iii) the 
conservative nature of some tandem repeats among different isolates. 
In this study molecular evidence is provided regarding the prevalence of A. marginale in 
eight of the nine South African provinces. Additionally, evolution of the genetic diversity 
of A. marginale msp1a in South Africa was studied, demonstrating that different codon 
positions of this gene evolved under positive or negative selection, likely due to immune 
selection and transmission fitness. Finally, these studies demonstrated the low 
variability of some tandem repeats commonly found among A. marginale strains. 
Collectively, results of this research will contribute toward development of new and 
novel vaccines for control of bovine anaplasmosis in South Africa and other regions of 
the world. 
45 
4.2 Materials and methods 
4.2.1 Study site and sample collection 
Blood samples were collected for these studies from May 2011 to July 2013 in 26 
districts and municipalities from eight South African provinces (Figure 5). Coordinates of 
the collection sites are provided in Table 4, as well as the farm production systems. 
Cattle for these studies were randomly selected and blood samples were collected only 
from adult animals after the owner's consent. While information regarding the age or 
sex of the animals was not recorded, vaccination histories were not available for these 
animals. Blood samples were collected by tail venupuncture using an 18 gauge needle 
and sterile 10 ml vacutainer EDTA tubes and stored at 4 °C. 
46 
- A. marginale epidemic areas 
- A. marginale endemic areas 
D Areas free of A. marginale 
~ Lesotho, area not included in the study 
Ticks species involved in A. margina/e transmission: + (R. evertsi everts1) 
* (R. microplus and R. decolaratus ) 
Figure 5: Map of South African areas included in the study. Map of South Africa showing the 
provinces that were included in the study (Limpopo, Mpumalanga, North West, Gauteng, KwaZulu-
Natal, Eastern Cape, Western Cape, and Northern Cape). Endemic, epidemic, and A. margina/e-free 
areas are coloured differentially (data collected from de Waal, 2000). The main tick species involved 
in the transmission of A. marginale in the sampled areas are shown: Rhipicephalus microplus, R. 
decoloratus, and R. evertsi evertsi (data collected from de Waal, 2000). 
47 
Table 4: Sampling sites and their coordinates in South African provinces. 
South African provinces Sampling sites Map coordinates 
Limpopo Makgodu, Masehlong, Chloen 23° 36'S, 29° 18'E, 
farm, Aganang Municipality 23° 32'S, 29° 03'E, 
23° 45'S, 28° 51 'E, 
Mpumalanga Ehlanzeni South District 25° 27'S, 30° 58'59"E 
Gauteng West Rand District, Merafong 26° 20' 1" S, 27° 19'39"E 
Municipality, Khutsong South and 26° 22'S, 27° 24'E 
Carltonville 
North West Moretele district, Maubane 25° 16'S, 28° 15'E 
KwaZulu-Natal Pietermaritzburg Chota, Umhlati 29°29'17.16"S, 
District, Albert Falls, Shallow drift, 30°26'27. 78"E, 
UMgungundlovu District, 29°28' 33.5" S, 
Richmond Municipality, Ndaleni 30°26' 11 .3" E 
Dip Tank 29° 52' 55.3" S, 
30° 40' 56.2" 
Eastern Cape Amathole District, Nkokobe 26°38'39"E, 
Municipality (Fort Beaufort, Alice) 32°46'52"S, 
and Middledrift and Nxuba 26°51 '25"E, 32'41 .12S, 
Municipality (Bedford, Adelaide) 27°11 '58"E, 
32° 52 '37"S, 
26°26'40"E, 
32°44'25"S, 
26° 18'18"E, 
32° 42'04"S 
Western Cape Stellenbosch district, Boland 33°44'28.6"S, 
18°59' 3"E 
Northern Cape John Taolo Gaetsewe District, 27°18'53"S, 
Ga-Segonyana Municipality, 23° 42 '15"E 
Kuruman, Zero Farm 
48 
4.2.2 DNA extraction 
Genomic DNA was extracted from cattle blood samples using ZR Genomic DNA r M 
Tissue Miniprep (Zymo Research, CA, USA). DNA was resuspended in DNA elution 
buffer and stored at -20 °C. The concentration of DNA was determined using the 
NanoDrop® ND-1000 (NanoDrop Technologies Inc., Wi lmington, USA). 
4.2.3 A. marginale species-specific PCR 
Specific set of primers were used to ampl ify msp 1a  1733F- (5'-
TGTGCTTATGGCAGACATTTCC-3'), 2957R- (5'- AAACCTTGTAGCCCCAACTTATCC-
3') - gene (Lew et al. , 2002). Primers were synthesized and supplied by lnqaba Biotech 
(lnqaba Biotechnical Industries (Pty) Ltd , Pretoria, Gauteng, South Africa). PCR 
reactions were prepared using Master Mix (Thermo Scientific DreamTaq Green PCR) , 
0.1-1 .0 µM of Forward and Reverse primers and 1 µg of DNA template in 25 µI final 
volume. The amplification cycles consisted of 40 cycles of 1 minute at 94 °C, 1 minute 
at 65 °C, and 2 minutes at 72 °C. Amplified products were separated in 1%  TBE (89 mM 
Tris-Borate, 2 mM EDTA, pH 8) agarose gel using 1 Kb ladder as a DNA size marker (1 
Kb DNA ladder, Life Sciences, Fermentas GmbH, Germany). DNA was visualized by 
gel staining in 0.5 µg/ml GelRed (Fermentas GmbH, Germany) under UV illumination 
and photographed. 
4.2.4 DNA sequencing of A. marginale msp1a gene 
PCR products containing only single amplification products of msp1a gene were 
sequenced at lnqaba Biotech facilities (lnqaba Biotechnical Industries (Pty) Ltd, 
Pretoria, Gauteng, South Africa). The termination reactions were performed using 
BigDye VER3.1 (ABI, Life Technologies, CA, USA) according to manufacturer's 
instructions. The labelled fragments were purified using Zymo research sequencing 
clean-up kit (Zymo Research, CA, USA) and subsequently analysed on a 3500xl 
Genetic Analyzer (ABI, Life Technologies, CA, USA). The sequences obtained in this 
study were submitted to GenBank and provided with accession numbers for msp1a 
(KC470153-KC470196) gene (Appendix 2). 
49 
4.2.5 Sequence analysis of A. marginale msp1a gene 
To identify the msp1a gene sequences obtained in our study the database Nucleotide 
collection (nr/nt) using Megablast (optimize for highly similar sequences) from the 
BLAST server was used (Zhang et al., 2000). Protein homology and identity analysis 
were performed using the multiple-alignment program ClustalW (Thompson et al., 
1994). The MSP1 a tandem repeats found in this study were reported previously 
(Cabezas-Cruz et al., 2013). 
4.2.6 Codon based phylogenetic analysis of tandem repeats 
Codon based alignment was performed using the codon suite server (Schneider et al. , 
2005; 2007). Detection of selection pressure on individual codons was calculated using 
two methods, single likelihood ancestor counting (SLAC) and fixed effects likelihood 
(FEL) (Pond and Frost. , 2005), used in the Datamonkey webserver (Delport et al., 2010; 
Pond and Frost. , 2005). Positive and negative selections were assigned to codon where 
w=dN (non-synonymous substitutions)/dS (synonymous substitutions) ratio was higher 
or lower than 1 respectively. The reconstruction of the ancestral amino acid sequence 
was performed using a neighbour joining tree rooted in tandem repeat 83 under the 
Dayhoff model of substitutions which was estimated to be the best model fitting the 
actual data. Three reconstruction methods were used: Joint (Pupko et al. , 2000), 
marginal (Yang et al., 1995) and sample (Nielsen, 2002) which are also used in the 
Datamonkey webserver. 
4.2.7 Amino acid variability, composition and genetic diversity index (GDI) 
The amino acid variability was calculated in the variability server (Garcia-Boronat et al. , 
2008) using the Shannon entropy (H) formula (Shannon, 1948) as fol low: 
M 
H = - LP;log2 P; 
i= I 
50 
Where Pi is the fraction of residues with a certain type of amino acid i, and M is the 
number of types of amino acid for a position. The proportion of variable over conserved 
positions was calculated as the number of positions with more than 0 of Shannon 
variability divided by the number of positions with 0 Shannon variability. Am ino acids 
were also classified regarding biochemistry properties, to know: negative charged, 
positively charged, uncharged-polar and non-polar. For comparison purposes, Shannon 
variability was additionally calculated in 28 and 43 MSP1 a sequences avai lable in 
GenBank from Venezuela and USA respectively. To further characterize the genetic 
diversity of MSP1 a, a genetic diversity index (GDI) was calculated for each A. marginale 
strain as follow: number of different MSP1 a tandem repeats divided by the total number 
of tandem repeats per strain . 1 and 0 were considered as maximum and minimum 
genetic diversity, respectively. 
Table 5: Observed prevalences of Anap/asma margina/e in different provinces of 
South Africa 
South African No. of blood Prevalence of A. No. of msp1a 
provinces samples collected marginale, sequenced 
per province msp1a-positive 
PCR (%) 
Limpopo 20 13(65) 8 
Mpumalanga 21 21 (100) 2 
Gauteng 20 20(100) 5 
North West 33 24(72) 7 
KwaZulu-Natal 55 50(90) 9 
Eastern Cape 40 40(100) 3 
Western Cape 16 14(87.5) 11 
Northern Cape 45 0(0) 0 
Free Statea 215 129(60) 29 
a Data collected by Mtshali et al. , (2007). 
51 
4.3 Results and discussion 
4.3.1 Molecular evidence of A. marginale prevalence in South Africa 
Bovine anaplasmosis has been considered to be endemic in South Africa, an 
assumption based primary on the distribution of the tick vectors (Ndou et al. , 2010) and 
the sero-prevalence of A. marginale which was determined only in the Free State 
(Dreyer et al., 1998), Limpopo (Rikhotso et al., 2005) and North West (Ndou et al. , 2010) 
provinces. Molecular evidence of endemic bovine anaplasmosis has not been reported 
in most of South Africa. 
This study was therefore designed to investigate the molecular evidence of A. marginale 
infection in cattle from Mpumalanga, Gauteng, Eastern Cape, Limpopo, North West, 
KwaZulu-Natal , Eastern Cape and Western Cape provinces (Table 5). Molecular 
diagnostic of anaplasmosis was determined previously in Free State province (Mtshali 
et al., 2007). In the present study, species-specific msp1a primers were used for PCR 
assays on 250 DNA samples obtained from cattle blood. 
The results of these PCR studies confirmed that A. marginale is widespread in South 
Africa, but with a variable prevalence in all the sampled provinces, except for Northern 
Cape in which no positive samples were detected (Table 5). The absence of A. 
marginale positive PCR in Northern Cape is not surprising because th is area is 
considered to be free of tick vectors (Mtshali and Mtshali, 2013) and the prevalence of 
Babesia spp., another tick-borne pathogen, was also found to be very low in this 
province (Mtshali and Mtshali , 2013). The provinces with highest prevalence of A. 
marginale were Mpumalanga, Gauteng, and Eastern Cape with an infection rate of 
100%. In the other provinces, A. marginale infections in cattle ranged from 65% to 90% 
(Table 5 and Figure 7). 
52 
Differences in the prevalence of A. marginale were not observed between commercial 
and communal farming systems. The prevalence of A. marginale in cattle from South 
Africa can be considered high when compared to the prevalence of A. margina/e in 
cattle from other regions of the world, for example, in Brazil a recent study found 70 % 
prevalence of A. marginale in a cattle herd (Pohl et al., 2013). Another study, by de la 
Fuente et al. (2005) showed a range from 25% to 100% of A. marginale prevalence in 
cattle herds from Italy. Analysis of the prevalence and genetic diversity of A. marginale 
MSP1 a in different geographic regions constitutes an important step toward 
development of effective MSP1 a based vaccines because the antigenic composition of 
the vaccine should contain MSP1 a variants present in different regions of the world . 
53 
A DDSSSASGQQQESSVSSQSE-ASTSSQLcr-
83 T ............ G.L ... C:;Q •• • ••• S.--
82 A •....•.•.•...• L •.. DLS .. W .... --
7 9 T • • • • • • • • P. • • • • Lr::;'f-,/ • DLS • • • . • . . - -
100 T . . . . . . • . . . . . G • L • . . GQ • • . . • • • • -
140 A ............ G.L ... GQ ••..•.• R-
141 T • . • • • • • . • • . . G . L • . . GQ • • • • • • • R-
142 T •..••••.••..•• IaP .• GH.R .... S.-
143 T • . • • G • • . • • . . • • L . . . SQ. RS . • . • . -
144 . . . . . . . . . . . . . . . IaP . . G(JJ) • • • •• s. -
145 G . . . . S • • . • • . . • • L • . . SQ . . . . . . . . -
146 A ..... ~ .......•.... - ........ -
147 T ..... ~ ........... G-.G ...... -
148 T ....• GD •.....•••.. G- •• A .•K .R-
149 T ••.•• @ ••••••••• L.G- ........ -
150 T ..... W .K ........ IG- ..... K .. -
151 AN'. . • . • •.• E . . . . L. . . DQ. . . . .... -
152 T . . . . . . . . . . . . . . L . . . DQ . . . . . . . R -
153 T . . . PE- . . . . . . . . F . . . AQ . • . . . . S . -
154 A . . . . . . . . . . . . . . L . . . DQ . . . . . . S . -
155 AN' • • • • • • • • • • • • • L . . . GQ • • • • • • • • -
158 T .. W. .......... L ...D Q ........ -
160 A • • • • • • • • • • • • • • P • • • • - • A. • • • • AD 
161 ~ ............ L •.. DQ •••.•... -
Figure 6: Newly reported sequences of MSP1 a tandem repeats. The one-letter amino 
acid code was used to depict MSP1 a repeat sequences. Dots indicate identical amino 
acids, and gaps indicate deletion/insertions. The ID of each repeat form was assigned 
previously in Cabezas-Cruz et al. , (2013). Tandem repeat A was used as a model for 
amino acid comparison. 
54 
4.3.2 A. marginale prevalence and msp1a genetic diversity 
The sequence of A. marginale msp1a was analysed in 44 strains (Table 4). We found 
52 different types of tandem repeats among South African strains (including those 
reported by Mtshali et al. , 2007 from Free State province) from which 23 were described 
for first time (Figure 6). Using a genetic diversity index (GDI), the genetic diversity per A. 
marginale strain we described (Table 6) and the average of genetic diversity was 
calculated per Province. A polynomial correlation (R2=0.7 6) occurred between the GDI 
and the prevalence of anaplasmosis per province (Figure 7). Interestingly, provinces 
with 100 % of A. margina/e prevalence were not the provinces with the highest or lowest 
GDI , rather these provinces were between a range of genetic diversity (0.82 - 0.87). 
Immune selection and transmission fitness have been suggested to impact genetic 
diversification of A. marginale (Palmer and Brayton, 2013). Immune pressure in cattle 
may induce greater genetic diversity in A. marginale populations in order to insure 
persistent infection. At the same time, the development of antigenic variation was 
suggested to have a transmission cost (Palmer and Brayton, 2013). A. marginale strains 
with lower transmission fitness due to high genetic variability would be at risk of 
elimination from the population (Palmer and Brayton, 2013). Furthermore, high 
incidence of ticks correlated to increased MSP1 a genetic variability in Argentina (Ruybal 
et al., 2009). 
These apparently contradictory aspects may be reconciled if increased tick transmission 
contributes to greater circulation of A. marginale strains in the cattle population, 
favouring the interaction between A. margina/e strains and potentially resistant hosts. In 
this study, the observed range of GDI in which 100% prevalence (an indicator of 
transmission efficiency) exist may reflect a positive balance between genetic diversity 
and biological transmission by ticks taking in account that, as shown in Figure 7, all the 
studied area, except for Northern Cape, in infected by ticks. 
55 
Notably, most of the new tandem repeats shown in Figure 6 were sequenced from 
KwaZulu-Natal province which has 90 % prevalence of A. marginale. When KwaZulu-
Natal was excluded from the correlation analysis between A. marginale prevalence and 
GDI (Figure 7), the polynomial regression increase from R2=0.76 to R2=0.98. A possible 
explanation of this result may be that the tandem repeats diversification found among 
KwaZulu-Natal MSP1 a provided a degree of transmission fitness to the strains present 
in that area. Another explanation could be that cattle immunity is increasing in the area, 
inducing A. marginale diversification for antigenic variation. One interesting question 
that remains unanswered is how the genetic diversity of A. marginale msp1a emerges in 
a specific region. 
4.3.3 Evolution of msp1a genetic diversity 
In order to analyse the evolution of msp1a genetic diversity observed in our samples, 
the tandem repeats (Table 6) were classified as "frequent" (present more than 22 times) 
or "rare" (present less than 10 times) based on the frequency of their appearance 
among South African A. marginale strains, including those from Free State province 
reported by Mtshali et al. (2007). The unique tandem repeats found in this study were 
classified as rare, being repeated, most of them, one time in only one strain (Figure 6). 
In contrast, tandem repeats 3, 4, 13, 34, Q and 37 had a high frequency (Table 6) and 
were also reported in A. marginale strains from Israel (3, 4), South America (4, 13) and 
Europe (Q). Repeat sequences 34 and 37 were abundant only in South Africa with rare 
exceptions (as reviewed by Cabezas-Cruz et al., 2013). Considering this, we wanted to 
test whether the tandem repeats newly described in this study (Figure 6) originated from 
extant A. marginale MSP1 a tandem repeats or had evolved from a tandem repeat that 
was lost after tandem repeat differentiation. 
56 
Table 6: Anaplasma margina/e strains and putative 20 structures of MSP1a tandem repeats 
A. marginale Province of Structure of msp1a GOia AVE-GDl/STDEV 
origin 
strain tandem repeats 
LP-7 Limpopo 34 159 0.917/0.144 
LP-10 Limpopo 27 13 3 36 
LP-30 Limpopo 27 13 3 
LP-34 Limpopo 34 13 3 38 
LP-37 Limpopo 27 13 13 37 0.75 
LP-46 Limpopo 3 38 
LP-50 Limpopo 34 13 13 0.667 
MP-C2 Mpumalanga 34 1 15 37 0.875/0.177 
MP-C5 Mpumalanga 15 15 100 83 0.75 
NW-C2 North West 27 13 4 4 37 0.8 0.960/0.089 
NW-C4 North West 27 13 4 37 
NW-C5 North West 82 13 79 4 37 
NW-C1-160312 North West 34 13 3 36 38 
NW-C4-160312 North West 34 36 38 3 
GP-C1 Gauteng 82 13 4 4 37 0.8 0.826/0 .173 
GP-C2 Gauteng 34 27 3 38 13 3 38 0.714 
GP-C5 Gauteng 3 4 4 4 37 0.6 
GP-C11 12105 Gauteng 34 37 
GP-C4117105 Gauteng 3 36 38 
GP-C7117105 Gauteng 34 13 13 0.667 
GP-C1817105 Gauteng 34 13 37 
KZN-D KwaZulu-Natal 42 43 25 163 31 0.919/0.128 
KZN-F KwaZulu-Natal 4 43 25 31 31 0.8 
KZN-K KwaZulu-Natal 27 13 4 4 37 0.8 
KZN-Y KwaZulu-Natal 143 144 145 146 
KZN-MM KwaZulu-Natal 42 43 25 31 
KZN-14 KwaZulu-Natal 142 43 25 31 
57 
KZN-19 KwaZulu-Natal 141 140 140 0.667 
KZN-49 KwaZulu-Natal 147 148 149 150 
KZN-51 KwaZulu-Natal 147 
EC-22 Eastern Cape 27 13 4 4 37 0.8 0.867/0.115 
EC-23 Eastern Cape 151 152 4 4 153 0.8 
EC-24 Eastern Cape 27 13 4 
WC-4 Western Cape 4 Q Q m 0.75 0.741 /0.286 
WC-6 Western Cape 3 4 4 37 0.75 
WC-7 Western Cape M M M M 0.25 
WC-8 Western Cape 34 4 37 
WC-10 Western Cape 154 
WC-11 Western Cape 40 Q Q Q Q Q 37 0.429 
WC-12 Western Cape 27 13 37 
WC-13 Western Cape M Q M Q M 0.4 
WC-14 Western Cape 155 36 38 
WC-15 Western Cape 161 13 37 4 162 
WC-16 Western Cape 34 13 4 13 13 4 37 0.571 
More common tandem repeats are highlighted (bold) and mentioned in order of abundance, from 
Higher to lower: 13,4,37 and 3, 34 have the same frequency, respectively. 
aGenetic diversity index of MSP1 a (GDI) was calculated as follows: number of different MSP1 a 
tandem repeats/total number of tandem repeats per strains. 1 is maximum genetic diversity. 
The average of GDI (AVE-GDI) and standard deviation (STDEV) for each region is shown. 
58 
120% 
Eastern Cape 
~--~ 
100% • • Mpumalanga 
G) 
u 
c • KwaZulu-Natal 
G) 
>"'  80% 
G) North West 
~ 
~ • • 
~ 60% • Free State 
Limpopo 
.as,  
e> 
ea,  40% 
20% 
y = 7387x4- 2534lx3 + 32490x2 -18449x + 3915.7 
R1 =0 .7675 
0% '--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-
0.7  0.75 0.8 0.85 0.9 0.95 1 
GDI 
Figure 7: Correlation between msp1a genetic diversity and A. marginale prevalence in South Africa. 
The prevalences of anaplasmosis in different South African provinces were plotted against the 
average of genetic diversity index (GDI). GDI was calculated for each strain as the number of 
different tandem repeats divided by the total number of tandem repeats. A polynomial correlation was 
found between these 2 parameters with R2 = 0.7 6. Provinces with 100% prevalence (Gauteng, 
Eastern Cape, and Mpumalanga) are in the range of 0.82-0.87 GDI whi le regions having less than 
100% prevalence are out of this range. 
59 
To test this hypothesis we reconstructed the ancestral state (Material and Methods) of 
the new tandem repeats presented in Figure 6. Surprisingly, the ancestral state of all 
the new tandem repeats was the tandem repeat 4 (Figure 8), suggesting that all of the 
newly described tandem repeats from South African A. marginale strains evolved from 
this tandem repeat. In addition, this evidence suggests that these repeated sequences 
may constitute a group of recently evolved tandem repeats specific to South Africa and, 
in fact, these repeats have not been reported elsewhere (Cabezas-Cruz et al., 2013). 
The mechanism for the generation of genetic diversity of A. marginale could be gene 
duplication followed by either mutation or mismatch repair as proposed by Palmer and 
Brayton. (2013). The variab le number of tandem repeats found among MSP1 a isolates 
suggests that tandem repeat duplication due to homologous recombination may occur 
in order to give a "substrate" for "testing" competitive advantage of new tandem repeats 
variants. As demonstrated by this research, new tandem repeats shown in Figure 6 
evolved from an initial tandem repeat 4 which served as a template for genetic variation. 
To test whether tandem repeat 4 evolved to the new forms under selective pressures, 
the ratio w (see Materials and Methods) was calculated for each codon position of the 
new tandem repeats from South Africa. We found that the diversification of tandem 
repeat 4 in South African strains occurred under both positive and negative selective 
pressures (Table 7 and Figure 8). Surprisingly, the positions evolving under negative 
selection (Figure 8 negative signs), 8 and 10, were reported before to be present in an 
immunodominant B-cell epitope present in MSP1 a (Figure 8, first boxed area, Garcia-
Garcia et al. , 2004) and the position 25, also evolving under negative selection, was 
found previously in a neutralization-sensitive epitope (Figure 8, second boxed area; 
Palmer et al., 1987; Allred et al., 1990). In contrast, one of the positions found to be 
evolving under positive selection was the amino acid position 20 (Figure 8, arrow) which 
has been implicated in the binding of MSP1 a to tick cells extract (de la Fuente et al., 
2003a). 
60 
These findings support the hypothesis discussed above that immune escape and tick 
transmission are both driving forces of A. marginale MSP1 a genetic diversification and 
suggest that tick transmission and immune escape would be triggers of the observed 
diversification of tandem repeat 4 in South Africa. This trend of purify ing 
selection/negative selection (or deletion of the unfit) for sites involved in immune 
recogn ition by the host was confirmed, in wider frame, by the following data: (i) most of 
the residues of the MSP1 a immunodominant B-cell epitope (Garcia-Garcia et al., 2004) 
were deleted in the tandem repeats forms a and 108: the tandem repeat a is 
widespread in strains from Mexico, Brazil , Venezuela, Argentina and Taiwan and is also 
present in the most common A. marginale strain of the world which have the tandem 
repeat composition {a, ~ . ~. ~. r ) (Cabezas-Cruz et al., 2013), and (ii) the first glutamine 
(Q) of the neutralization-sensitive epitope (Palmer et al., 1987; Allred et al., 1990) is 
deleted in several tandem repeats {A, D, E, y, L, q> , 5 - 9, 14, 31 , 36, 52, 57, 60 - 66, 69 
- 72, 76, 84- 86, 95 - 99, 105, 107, 116-119, 129-131 , 136 - 139) from A. marginale 
strains reported previously (Cabezas-Cruz et al. , 201 3). As seen in Figure 8, Q was 
deleted in some of South African new tandem repeats (146, 147, 148, 149 and 150). 
Collectively, this evidence suggests that purifying selection is most likely one of the 
mechanisms that A. marginale had evolved to escape immune recognition toward 
MSP1a. 
Tandem repeat 4 from MSP1a was also found in A. marginale strains isolated from an 
anaplasmosis outbreak in Mexico where R. microplus was implicated as tick vector 
(Almazan et al., 2008), and new tandem repeats were reported also in th is study. It 
should be interesting to test whether these newly described Mexican MSP1 a variants 
evolved from tandem repeat 4. We consider these findings to be relevant to the 
development of MSP1 a based vaccines and suggests that MSP1 a based vaccination 
should be combined with tick control strategies in order to minimize genetic diversity of 
A. marginale msp1a. A recent study was reported that combined use of a tick protective 
antigen with MSP1 a in the same vaccine formulation that was directed toward control 
both tick infestations and anaplasmosis (Torina et al., 2014). 
61 
Table 7:Sites that evolved under positive and negative selection in the new 
tandem repeats from South Africa 
Codons Methods SLAC Methods FEL Type of 
selection 
w(dN/dS p value w(dN/dS p value 
1 5.49 0.043 Infinite 0.012 Positive 
6 1.58 0.311 Infinite 0.093 Positive 
16 1.39 0.371 Infinite 0.196 Positive 
20 2.86 0.172 Infinite 0.133 Positive 
28 2.22 0.197 Infinite 0.079 Positive 
8 -1 .66 0.373 -5.14 0.236 Negative 
10 -6.81 0.037 -13.89 0.016 Negative 
25 -3.57 0.134 -6.56 0.110 Negative 
62 
~ 
+ + -- + + - + 
-------------1.. --160 ANG1SSASGQQQESSWLsQSDQASTSSQLG 149 TDSSSAGDQQQESSVSSLSG-ASTSSQLG 
__, .. ·-------145 GDSSSSSGQQQESSVLSQSSQASTSSQLG 
-.--158 AESSSASGQQQESSVLSQSDQ-STSSQLG 
__, ...--.... 148 TDSSSAGDQQQESSVSSQSG-ASASSKLR 
·-----142 TDSSSASGQQQESSVLPQSGBARTSSQSG 
.---100 TDSSSASGQQQESGVLSQSGQASTSSQLG 
---------....... 161 ADSSSASGQQQESSVLSQSDEASTSSQLG 
_____. ...,._. ---18421  TDSSSASGQQQESGVLSQSGQASTSSQLR 
ADSSSASGQQQESSVLSQSDLSSTWSQLG 
-.--154 ADSSSASGQQQESSVLSQSDQASTSSQSG 
__, ...--.... 150 TDSSSAGDQKQESSVSSQXG-ASTSSKLG 
·-----143 TDSSSGSGQQQESSVLSQSSQARSSSQLG 
.---151 ANSSSASGQQEESSVLSQSDQASTSSQLG 
--------~··-r------'--1--41447  TDSSSAGNQQQESSVSSQSG-AGTSSQLG DDSSSASGQQQESSVLPQSGQX>STSSQSG • .---140 ADSSSASGQQQESGVLSQSGQASTSSQLR __, ...--.... 79 TDSSSASGQPQESSVLCVSDLSSTSSQLG 
I -1 ----153 TDSSPEMGQQQESSVFSQSAQASTSSQSG 
·--------155 ANSSSASGQQQESSVLSQSGQASTSSQLG 
-------------------152 TDSSSASGQQQESSVLSQSDQASTSSQLR 
1,_,_ --_-_-_-_-_--_-_-_-_-_--_-_-_-_-_--_-_-_-_1_4 836  ADSSSAGNQQQESSVSSQSE-ASTSSQLG 
TDSSSASGQQQESGVLSQSGQASTSSQSG 
~R 4 : TDSSSASGQQQESSVLSQSG~STSSQLG • 
Figure 8: Reconstruction of ancestral amino acid sequence and amino acid positions under positive and 
negative selection. The reconstruction of the ancestral state (TR 4: Tandem repeat 4, sequence marked 
with *) of the new tandem repeats found in South Africa (Figure 6) was performed using 3 reconstruction 
methods, namely: joint, marginal, and sample (see 'Materials and methods' for details). Positions that 
evolve under negative (-) and positive (+) selection are shown (see Table 7). Amino acid at position 20 is 
indicated (arrow). The residues of the immunodominant 8-cell epitope (Garcia-Garcia et al., 2004) (first 
box) and the neutralization-sensitive epitope (Palmer et al., 1987; Allred et al., 1990) (second box) are also 
shown. 
63 
4.3.4 Amino acid variability and low variable msp1a peptides 
Genetic variability could also be analysed by calculating the amino acid variability in 
each position of the tandem repeat. In order to compare the msp1a genetic variability in 
South Africa with other regions of the world we calculated the amino acid variability for 
29 amino acid positions from all the msp1a tandem repeats found in South Africa, USA 
and Venezuela which were available in GenBank and collected by Cabezas-Cruz et al., 
2013. 
The amino acid variability of msp1a tandem repeats from South Africa can be seen in 
Figure 9 as compared with that from USA (low) and Venezuela (high). In agreement 
with th is, the average of amino acid variability was 0.30, 0.42 and 0.7 2 for USA, South 
Africa and Venezuela, respectively (Figure 9A-C). In comparison the proportion of 
variable over conserved positions in MSP1a tandem repeats was higher Venezuela (29) 
compared to USA (0.85) and South Africa (2.2). Epitopes from MSP1 a were found to 
induce a protective immune response against A. marginale (Santos et al. , 2013). 
Considering this, using the variability server (Garcia-Boronat et al., 2008), we explored 
whether some low variable peptides could be found in MSP1 a from South Africa, USA 
and Venezuela. We observed that the amino acid variability of MSP1a  tandem repeats 
from South Africa and USA supported the existence of low variable peptides (Figure 9) 
which may be useful in development of peptide-based MSP1 a vaccines, while the high 
amino variability of MSP1 a tandem repeats from Venezuela do not support for this type 
of low variable peptide. Interestingly, the low variability peptide in South Africa overlap 
with the position of the immunodominant B-cell epitope reported previously for MSP1 a 
(Garcia-Garcia et al., 2004). 
64 
Venezuela Av.rage of' amino acids ~rlablllty: 0 .72 
Proportion ot' vartable/cons..-"9d sit-: 29 
3 
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2 4 25 26 27 28 29 
•Negative • Positive m Uncharged- polar • Nonpolar a Deleted 
USA Av.r3ge of' amino acids ~rlablltty: 0.30 
3 Proportion of' vartablelcons..-"9d sit-: 0 . 85 
~ 
1-i -c: 2 ,.... c: 
-<c>:  ., c: ~ U> 
0 ., 2 3 4 5 6 7 8 9 .,0 .,., .,2 .,3 14 ., 5 16 17 18 19 20 21 22 23 2 4 25 26 27 28 29 
G Q Q Q E S S V SS Q s* 
-Negative • Positiv e 1:11 Uncharge d -polar - Nonpolar o Oeleted 
South Af'rfc.a Av.r3ge of' amino acids ~rlablllty: 0 .42 
3 Proportion of' vartable/conser\19d sit-: 2.2 
~ 
1i 2 
1i 
> 
c: 
-2 ., c: ~ U> 
0 ., 2 3 4 5 6 7 8 9 '10 1., 1 2 1 3 14 15 ., 6 17 18 19 2 0 2 1 22 23 2 4 25 26 27 28 29 
sis s As o o o o E s slv * 
•Negativ e • Positive £J Uncha rged- polar • Nonpola r CJ Deleted 
Figure 9: MSP1a amino acid variability, composition. and low-variable peptides. The figure shows 
the amino acid variability and composition among the tandem repeats in 3 different countries: 
Venezuela (A). USA (B), and South Africa (C). Venezuela shows a high proportion of 
variable/conserved sites and a high average of amino acid variability while South Africa and the 
USA show middle and lower values, respectively. Different colours in columns depict different 
biochemical properties in the amino acid composition: negative (green). positive (red), uncharged-
polar (beige), and non-polar (blue); proportion of deleted positions is shown in yellow. Consensus 
sequences of low-variable peptides are shown (*) for the USA and South Africa. The region of the 
immunodominant B-cell epitope from A. marginale (Garcia-Garcia et al., 2004) is boxed in the low-
variable peptide from South Africa. (For interpretation of the references to colour in this figure 
legend, the reader is referred to the web version of this article.) 
65 
CHAPTER 5 
STRUCTURAL AND PHYLOGENETIC ANALYSES OF ANAPLASMA MARG/NALE 
INFECTING CATTLE IN SOUTH AFRICA USING msp1a AND msp4 GENES 
5.1 Introduction 
Phylogenetic analysis establishes the relationships between genes or gene fragments, 
by inferring the common history of the genes or their fragments. To achieve this, it is 
essential that homologous sites be compared with each other (positional homology) 
(Nei and Kumar, 2000). Phylogenetic studies have been previously conducted to 
resolve and understand the Anaplasma species and how these species relate to one 
another (de la Fuente et al. , 2007a). 
The phylogenetic relationship and evolution of A. marginale isolates is important for 
understanding the biology and the possibilities for control of anaplasmosis (Kocan et al., 
2002). Recent research has focused on MSPs that are involved in interactions with 
vertebrate and invertebrate host cells (McGarey and Allred, 1994; McGarey et al. , 1994 
de la Fuente et al., 2001 b) and have been used to elucidate the phylogeographic 
patterns of A. marginale (Kano et al., 2002; Lew et al., 2002; de la Fuente et al., 2007a). 
MSP1 a and MSP4 genes were used previously, for phylogenetic analysis that was 
found to be involved in interactions with both vertebrate and invertebrate hosts 
(McGarey and Allred, 1994; McGarey et al. , 1994; de la Fuente et al. , 2001b). 
Therefore, these MSPs are also likely to evolve more rapidly than other nuclear genes 
because they are subjected to selective pressures exerted by host immune systems. 
66 
Both msp1a and msp4 genes have been found to be stable genetic markers during 
multipl ication of A. marginale (de la Fuente et al., 2001a; Bowie et al., 2002). MSP1a 
was also shown to effect transmission of A. marginale by ticks (de la Fuente et al. , 
2001 a; Kocan et al. , 2010). These genes were reported to be more stable for 
phylogenetic studies because the MSPs do not appear to undergo antigenic variation in 
cattle or ticks (Bowie et al., 2002). MSP1 a, encoded by msp1a, has been reported to be 
an adhesin for bovine erythrocytes and tick cells and to effect adhesin, infection, and 
transmission of A. marginale by ticks of the genus Dermacentor (McGarey and Allred , 
1994; McGarey et al., 1994; de la Fuente et al., 2001 c). Although the specific function of 
MSP4 is not known, previous analysis of the msp4 gene from A. marginale isolates 
demonstrated that its sequence varies sufficiently among isolates to support its use in 
phylogeographic studies (de la Fuente et al. , 2003a). In South Africa, phylogenetic 
profile of A. marginale infecting cattle has not been determined meaning that the genetic 
relationship between South Africa A. marginale isolates and those in other African 
countries is unknown. Therefore, the aim of this study is to establish the phylogenetic 
profile of A. marginale infecting South Africa cattle using msp1a and msp4 as marker 
genes. 
5.2 Materials and methods 
5.2.1 DNA extraction 
The genomic DNA was extracted from cattle blood samples using ZR Genomic DNA TM 
Tissue Miniprep (Zymo Research, CA, USA). Total volume of blood sample was 
adjusted in a microcentrifuge tube with water to 100 µI prior to adding 95 µI of 2x 
Digestion buffer and 5 µI of Proteinase K. The mixture was thoroughly mixed and then 
incubated at 55 °C for 20 minutes. A volume of 700 µI of genomic lysis buffer was 
added to the tube and mixed thoroughly by vortexing. 
67 
The mixture was carefully transferred to a Zymo-Spin ™ llC column in a collection tube 
and centrifuged at 10,000 x g for one minute. A volume of 200 µI of DNA pre-wash 
buffer was added to the spin column in a new collection tube and centrifuged at 10,000 
x g for one minute. A volume of 400 µI of g-DNA wash buffer was added to the spin 
column and centrifuged at 10, 000 x g for one minute. The spin columns were carefully 
transferred to a clean 1.5 ml microcentrifuge tube and 100 µI of DNA Elution buffer 
added to the spin column. The microcentrifuge tube was incubated for 5 minutes at 
room temperature, and then centrifuged at 8000 rpm for 30 seconds to elute the DNA. 
The eluted DNA was stored at -20 °C until used for studies. The concentration of DNA 
was measured using a NanoDrop® ND-1000 (NanoDrop Technologies Inc., Wilmington, 
USA). 
5.2.2 Set of primers used for amplification of msp1a and msp4 genes 
Table 8: Oligonucleotide sequence of primers used in this study and their references 
Primer Oligonucleotide (5'-3') Reference 
designation 
1733F TGTGCTTATGGCAGACATTTCC Lew et al., 2002 
2957R AAACCTTGTAGCCCCAACTTATCC Lew et al., 2002 
MSP45F GGGAGCTCCTATGAATTACAGAGAATTGTTTAC de la Fuente et al., 2003a 
MSP45R AAGCTCGAGGCTGAACAGGAATCTTGCTCCAAG de la Fuente et al. , 2003a 
68 
5.2.3 Anaplasma marginale species-specific PCR of the msp1a gene 
Specific set of primers were used to amplify msp1a gene (Table 8). Primers were 
synthesized and supplied by lnqaba Biotech (lnqaba Biotechnical Industries (Pty) Ltd, 
Pretoria, Gauteng, South Africa). The PCR reagents mixture consisted of total volume 
of 25 µI of Thermo Scientific DreamTaq Green PCR Master Mix (2X), 10 µM of forward 
and reverse primers, 1 µg Template DNA in 0.2 ml PCR tubes. The PCR mixture was 
then amplified by DNA thermocycler (Bio-Rad T100™ DNA Thermal Cycler, Bio-Rad 
Laboratories, Johannesburg, South Africa). The amplification process involved an initial 
denaturation at 94 °C for 3 minutes followed by 40 cycles of denaturation at 94 °C for 1 
minute at 94 °C, annealing at 65 °C for 1 minute, and an extension at 72 °C for 2 
minutes. A cycle of a final extension was done at 72 °C for 7 minutes. The amplified 
products were then separated by electrophoresis using 1%  agarose gel immersed in 
TBE buffer (89 mM Tris-Borate, 2 mM EDTA, pH 8). One kilo-base pair ladder marker 
was used to determine the size of the PCR products after staining with 0.5 µg/ml 
GelRed (Life Sciences, Fermentas GmbH, Germany) and visualization under UV 
illumination. Gel documentation was done and taking a photograph using a camera. 
5.2.4 Anaplasma. marginale species-specific PCR of the msp4 gene 
The msp4 gene was amplified from 5 µI per 20 µI reaction volume using 10 pmol of 
each primer (Table 7) (2x Phusion Flash PCR Master Mix) (Thermo Fisher Scientific, 
USA). Reactions were performed in a BIO-RAD T100™ DNA thermal cycler (Bio-Rad 
Laboratories, Johannesburg, South Africa). The amplification cycles following an initial 
denaturation at 98 °C for 3 minutes for 1 cycle then consisted of 3-step protocol of a 
denaturation step of 98 °C for 10 seconds, annealing at 63 °C for 30 second and 
extension at 72 °C for 30 seconds for 5 cycles, 98 °C for 1O  seconds, annealing at 65 °C 
for 30 seconds and extension at 72 °C for 30 seconds for 5 cycles, 98 °C for 10 
seconds, annealing at 67 °C for 30 seconds and extension at 72 °C for 30 seconds for 
20 cycles, with a final cycle at 72 °C for 7 minutes. 
69 
The ampl ified products were then separated by electrophoresis using 1 %agarose gel 
immersed in TBE (89 mm Tris-Borate, 2 mm EDTA, pH 8). One kilo-base pair ladder 
marker was used to determine the size of the PCR products after staining with 0.5 µg/ml 
GelRed (Life Sciences, Fermentas GmbH, Germany) and visualization under UV 
illumination. Gel documentation was done and taking a photograph using a camera 
(Figure 10). 
70 
M 2 3 4 5 6 7 8 
1000 
900 
800 
700 
600 
500 
400 
300 
200 
100 
Figure 10: Gel image of 1%  agarose gel electrophoresis of PCR amplification products 
obtained from A. marginale from Western Cape Province in South Africa using primer 
MSP45F and MSP45R. Lane M: GeneRuler™ 1 Kb DNA ladder, ready-to-use; Lane 1- 7 
indicate positive results and lane 8 indicate negative control. 
71 
5.2.5 DNA sequence alignment and phylogenetic analysis 
All A. marginale msp1a and msp4 gene sequences and GenBank accessions utilised 
here are listed in Appendix 2, 3 and 4. The obtained sequences were edited using 
Chromas V1 .49 software and BioEdit (Tom Hall Ibis Biosciences, Carlsbad , USA). 
Nucleotides were coded as unordered, discrete characters with five possible character 
states: A, C, G, T or N and gaps were coded as missing data. The percent identity 
between the tandem repeat in position 1 (R1) and the other tandem repeats was 
determined using CLUSTALW. The tandem repeats secondary structure was predicted 
using the PSIPRED server (Buchan et al., 2010). 
The sequences obtained in this study were compared with available sequences from 
the NCBI database using the Basic Local Alignment Search Tool (BLAST) 
(http://www.ncbi.nlm.nih.gov/BLAST/). The sequences were submitted to GenBank 
(Appendix 3). Multiple sequence alignment was performed using ClustalW 1.6 program 
(Thompson et al., 1994). Phylogenetic analysis was conducted in MEGA 5 (Tamura et 
al. , 2007) using Neighbour joining (NJ) and Maximum Likelihood (ML) algorithm (Saitou 
and Nei, 1987). The evolutionary distances were computed using with Kimura 2-
parameters correction (Kimura, 1980) with bootstrap analysis of 1000 replicates. 
Sequences of A. marginale msp4 reported previously from West, East and North Africa, 
North and South America, Europe, Asia and Australia were obtained from GenBank and 
used for the msp4 gene phylogenetic analysis of South African isolates sequences 
(Appendix 3 and 4). The A. ovis and A. phagocytophilum (GenBank accession number 
AF393742 and AF530197 respectively) were used as outgroups. 
72 
A total of 5 phylogenetic trees were constructed to determine the relatedness of South 
African A. marginale strains with strains from West, East and North Africa, North and 
South America, Europe, Asia and Australia (Appendix 4). One phylogenetic tree was 
constructed based on the msp1a gene sequences using the maximum likelihood 
method (Figure 12). Two phylogenetic trees were constructed based on msp4 gene 
sequences using neighbor- joining method and the other two using maximum-likelihood 
method (Figure 13a and 14a). One phylogenetic tree for each neighbor-joining and 
maximum likel ihood method was constructed from South African province isolates and 
the other with South African province isolates together with the sequences obtained 
from West, East and North Africa, North and South America, Europe, Asia and Australia 
the obtained from the GenBank (Figure 13a and 13b) (Figure 14a and 14b) (Appendix 
4). Anaplasma ovis and A. phagocytophilum were used as outgroups for the 
construction of phylogenetic tree based on msp4 gene sequences. 
5.3 Results 
Previous studies revealed that the gene and protein sequences of msp1a and msp4 
nucleotide sequences have been used to infer the phylogenetic relationships among 
Oklahoma and New World isolates from Argentina, Brazil, Mexico and the United 
States. All the 11 A. marginale isolates collected from Oklahoma had diffe rent msp 1a  
sequences but identical to msp4 sequences (de la Fuente et al., 2003a). Phylogenetic 
studies of South African isolates using msp4 gene sequences demonstrated that A. 
marginale isolates had a 100% and 99% identity with the sequences from West, East 
and North Africa, North and South America, Europe, Asia and Australia sequences 
available in the GenBank (Appendix 4). 
73 
5.3.1 Phylogenetic analysis of A. marginale isolates using msp1a gene 
5.3.1.1 MSP1 a sequence analysis 
The msp1a sequences of 44 A. marginale isolates were analysed as shown in Table 6. 
Differences were found in the tandem repeat sequences and structure of msp1a genes 
among the isolates included in th is study. The msp1a tandem repeats gave 23 new 
sequences with amino acid changes (Figure 6) resulting in identification of forty four 
different strains of A. marginale (Table 6). The strain containing the tandem repeat 
structure 27/13/4/4/37 was found twice as KZN-K (Table 6). Tandem repeats 3, 13, 27, 
34 and 37 were common in the study areas in all provinces and tandem repeat 42 and 
43 were highly represented in KwaZulu-Natal (Table 6). 
A pattern of proportional decrease in the % of homology of R1 with the subsequent 
tandem repeats (R1 + n) was obtained. The % of homology between R1 and the 
tandem repeat in position (R1 + n) will have a decrease proportional to the % of 
homology between R1 and the tandem repeat in position (R1 + 1) (Figure 11 ). 
Differential pattern of proportional decrease in amino acid homology was found in 
KwaZulu-Natal (Figure 11 ). On the other hand, the percent of homology differed among 
the tandem repeats in the provinces in Figure 11 a (approximately 90% and 70% 
respectively). 
74 
South African 
Identity among tandem repeats 
A provinces with this pattern 
96% 
R1 Limpopo Mpumalanga 
93% North West Eastern Cape 
11 
89% Gauteng Western Cape 
B 
89% 
R1 KwaZulu-Natal 
7<:1/'o 
11 
42% 
Figure 11: Proportional identity between tandem repeat in position R 1 and R 1+ n. 
This pattern was used to suggest a flow of transmission/migration from Eastern Cape to 
KwaZulu-Natal. The strain containing the tandem repeat structure 27/1 3/4/4/37 was 
found twice, once in KwaZulu-Natal and then again in the Eastern Cape (Figure 12). 
Taking in account that this strain does not follow the pattern of proportional decrease 
found in KwaZulu-Natal, it was concluded that th is strain was introduced from Eastern 
Cape and this conclusion was in agreement with the maximum likelihood phylogenetic 
analysis of strains found in KwaZulu-Natal, where KZN-K (27/13/4/4/37) was the only 
one apart the cluster formed by the other strains from that province (Figure 12). 
75 
Table 9: Anaplasma marginale strains and putative 20 structures of MSP1a tandem repeats 
A. marginale Province of Structure of msp1a MSP1a tandem 
origin 
strain tandem repeats repeats 20 structure 
LP-7 Limpopo 34 15 (a-a) (a-a) 
LP-10 Limpopo 27 13 3 36 (a-a)(a-a)(a-c)(c-a) 
LP-30 Limpopo 27 13 3 (a-a)(a-a)(a-c) 
LP-34 Limpopo 34 13 3 38 (a-a)(a-a)(a-c)(a-c) 
LP-37 Limpopo 27 13 13 37 (a-a)(a-a)(a-a)(a-c) 
LP-46 Limpopo 3 38 (a-c)(a-c) 
LP-SO Limpopo 34 13 13 (a-a)(a-a)(a-a) 
MP-C2 Mpumalanga 34 13 1S8 37 (a-a)( a-a) (a-a)(a-c) 
MP-CS Mpumalanga 1S 1S 100 83 (a-c)(a-c)(a-c)( a-c) 
NW-C2 North West 27 13 4 4 37 (a-a)( a-a)( a-13)( a-13)( a-c) 
NW-C4 North West 27 13 4 37 (a-a)( a-a)( a-13 )( a-c ) 
NW-CS North West 82 13 79 4 37 ( a-c)( a-a)( a-a)(a-13 )( a-c) 
NW-C 1-16031 2 North West 3 13 3 36 38 (a-a)( a-a)( a-c)( c-a )(a-c) 
NW-C4-160312 North West 34 36 38 3 (a-a )( c-a)(a-c)( a-c) 
GP-C1 Gauteng 82 13 4 4 37 ( a-c )(a-a)( a-13 )( a-13 )( a-c) 
GP-C2 Gauteng 34 27 3 38 13 3 38 (a-a )(a-a)( a-c)(a-c)( a-a) (a -c)( a-c) 
GP-CS Gauteng 3 4 4 4 37 (a -c )( a-13 )( a-13 )( a-13 )(a -c ) 
GP-C111210S Gauteng 34 37 (a-a)(a-c) 
GP-C411710S Gauteng 3 36 38 (a-c)(c-a)(a-c) 
GP-C711710S Gauteng 34 13 13 (a-a)(a-c)(a-c ) 
GP-C18171 OS Gauteng 34 13 37 (a-a)(a-c)(a-c) 
KZN-D KwaZulu-Natal 42 43 2 1 3 31 (a-c)(a-c)(a-c) (a-a)(a-a) 
KZN-F KwaZulu-Natal 4 43 2S 31 31 (a-c)( a-c)( a-c)( a-a)( a-a) 
KZN-K KwaZulu-Natal 27 13 4 37 (a-a) (a-a) (a-13)(a-13)(a-c) 
KZN-Y KwaZulu-Natal 143 144 14S 146 ( aa)( a-c )( a-c)( a-a ) 
KZN-MM KwaZulu-Natal 42 43 2S 31 (a-c)(a-c)(a -c)( a-a) 
KZN-14 KwaZulu-Natal 142 43 2S 31 (c)(a-c)(a-c)(a-a) 
KZN-19 KwaZulu-Natal 141 140 140 (a-c)(l3-c)(l3-c) 
KZN-49 KwaZulu-Natal 147 148 149 1 0 (a-c)(a-c)(a-c)(a-c) 
KZN-S1 KwaZulu-Natal 147 (a-c) 
EC-22 Eastern Cape 27 13 4 3 (a-a )( a-a)( a-13)( a -13)( a-c) 
EC-23 Eastern Cape 1 S1 1 2 4 4 1S3 (o -o )(o-a-a )(o -13)( o -13 )( oo) 
EC-24 Eastern Cape 27 13 4 (a-a)(a-a) (a-13) 
WC-4 Western Cape 4 Q Q m (o-a )(a-a)(o-a)(a-c) 
WC-Q Western Cape 3 4 4 37 (a-c)( a-13)(a-13)(a-c) 
WC-7 Western Cape M M M M (a-c)( a-c)(a -c)(a -c ) 
76 
WC-8 Western Cape 34 4 37 (a-a)(a-~)(a-c) 
WC-10 Western Cape 154 (a-c) 
WC-11 Western Cape 40 Q Q Q Q Q 37 (a-a)(a-a)(a-a)(a-a )(a-a) (a-a)(a-c) 
WC-12 Western Cape 27 13 37 (a-a)(a-a)(a-c) 
WC-13 Western Cape M Q M Q M (a-c)(a-a)(a-c)(a-o) (a-c) 
WC-14 Western Cape 155 36 38 (aa)(c-a)(a-c) 
WC-15 Western Cape 161 13 37 4 162 (a-a)( a-a)( a-c)( a-~ )(a-a) 
WC-16 Western Cape 34 13 4 13 13 4 37 (a-o)(a-a )(a-~)( a-a)( a-a) (a-~)( a-c) 
The A. marginale strains detected in this study are presented as msp1a tandem repeats 
organization. Limpopo (LP), Mpumalanga (MP), North West (NW), Gauteng (GP), 
KwaZulu-Natal (KZN), Eastern Cape (EC) and Western Cape (WC). The code-numbers 
for tandem repeats are shown in Figure 2. The putative secondary structure of every 
tandem repeat is shown as two separated alpha helices (a-a), one long alpha helix (aa), 
beta strand (13) and coiled structure (c). 
The pattern found in LP, MP, NW, GP, KZN, EC and WC provinces is listed (Figure 11 ); 
reductions of approximately 3% were observed among A. marginale msp1a tandem 
repeats. The A. marginale strain NW-C4 served as a model (A). Sequences from 
KwaZulu-Natal did not follow this pattern and reductions of approximately 10% and 40% 
were observed. The A. marginale strain KZN-14 served as a model (B). 
77 
A . marutnale KZM-51 
0 .06 
Figure 12: Maximum- likelihood phylogenetic tree based on the MSP1 a from the strains 
present in KwaZulu-Natal and North West provinces. In this tree the strain A. marginale 
KZN-K (red dot) was in the cluster from North West sequences (blue circle). Bootstrap 
values are shown as a percentage in the internal branch. Only bootstrap values higher 
than 50% were shown. NJ phylogenetic analysis gave similar results (Data not shown). 
78 
5.3.2 Phylogenetic analysis using msp4 gene sequences 
The msp4 gene of A. marginale isolates from Limpopo, Mpumalanga, North West, Gauteng, 
KwaZulu-Natal , Eastern Cape, Northern Cape and Western Cape provinces of South Africa 
were amplified by PCR and then sequenced (Figure 10). Positive results were not obtained 
from Northern Cape samples. Two samples from each province were randomly selected for 
phylogenetic analysis. Four phylogenetic trees were each constructed for A. marginale isolates 
randomly selected from the LP, MP, GP, NW, GP, KZN, EC and WC provinces of South Africa 
(Figure 13a and 14a) (Appendix 3), and those from the other countries in Africa and around the 
world (Figure 13b and 14b) (Appendix 4). Anaplasma ovis and A. phagocytophilum were 
included as outgroup taxa for the phylogenetic analysis based on the msp4 gene. 
5.3.3 Phylogenetic analysis of msp4 gene using neighbor -joining phylogenetic tree 
A complete msp4 gene was obtained after sequencing using the primers designed 
previously to amplify msp4 gene (Appendix 3). The A. marginale msp4 gene was amplified 
and sequenced isolates infecting cattle from LP, MP, NW, GP, KZN, EC and WC and the 
results revealed that this nucleotide differed in length and identity. 
Anaplasma marginale isolates from LP, GP, NW, KZN and WC had seven different 
nucleotides from MP and EC isolates at position 163, 197, 223, 239, 251 , 281 and 725 with 
the following changes A to G, G to A, C to A, G to A, A to G, G to A and G to A respectively 
(Table 10). These changes gave to two clades from South African isolates and were 
classified as South African first clade and South African second clade. The first South 
African clade consists of isolates from LP, GP, NW, KZN, and WC and the second South 
African clade consist of isolates from MP and EC (Figure 13a, 13b, 14a and 14b). The first 
clade had 99% bootstrap support and was the same as the second clade, thus 
demonstrating that South African A. marginale isolates are more closely genetically related. 
79 
Table 10: Differences in the A. marginale msp4 nucleotide sequences of 
LP,NW,GP,KZN,WC and MP, EC isolates of South Africa 
Nucleotide position 
Isolates 163 197 223 239 251 281 725 
LP A G c G A G G 
NW A G c G A G G 
GP A G c G A G G 
KZN A G c G A G G 
WC A G c G A G G 
MP G A A A G A A 
EC G A A A G A A 
In most of the A. marginale msp4 isolates that have been sequenced, a 849 bp nucleotides 
with coding sequences were amplified. 
80 
50 
Figure 13a: Neighbor-joining phylogenetic tree of A. marginale msp4 gene sequences from strains 
identified in cattle in South Africa. The phylogenetic tree was implemented in the MEGA5 (Tamura 
et al. , 2011 ). Bootstrap analysis was conducted with 1000 replicates. The Gen Bank accession 
numbers of the respective sequences used for the phylogenetic analysis are indicated at the 
beginning of the name of each sequence. 
81 
AF428084 1 Mexico 
AF428083 1 Mexico 
57 AF428085 1 Mexico Southern hemisphere 
AY283190 1 Brazil 4 
AY191827 1 Puerto Rico 
AF428086. 1 Argentina 
51 '------AY787172 1 lsraeh round 
65 o-- ----- -AY456002 1 SP7-Spa1n 
'----- - --- - JN572Q28 1 GZB.China 
'-- --------AY786994.1 Israeli tailed 
AY127076 1 S!Jllwater 2 
AY010253.1 Okeechobee 
56 AY127073.1 O klahoma City 
'----+--- AY010254 1 V1rg1n1a 
100 68
AY0 10251.1 M1&&1ss1ppt 
EU315782.1 50 (G16}-Hungary Northern hemisphere 
S3 EU315783 1 51 (G18}-Hungary 
AY702921 .1 na1y 47 
.-----s-2 t---- AY702917 1 na1y 6 
AY786993. 1 Israeli non-tailed 
100 
AF428081 .1 USA 
55 AY127065.1 Oregon 
AY010248.1 Calllomia 
S• AY127077 1 New Castle 
AY127075.1 S tillwater 1 
A Y 127068. 1 Glencoe 2 
'--- -------- - ----· AY706389 Anaplasma phagocy1ophilum 
'-------------------• AF39374 2 .1 Anaplas ma ollis 
.--------- ------ - AY;>83191 1 Brazil 5 
A Y666007. 1 2 .4-Zimbabwe 
~-7-1 o----AY666006.1 1 6-Zimbabwe 
AY666011 .1 5 .9-Zimbabwe 
100 '------AY666004.1 661Kan-Kenya 
90 
AY666003.1 G38-Australoa 
SS A Y666002.1 F72-Austraha 
.---- -· KF758867 MP-C15 
t----· KF758869 MP-C18 
81 
• KF758845 EC-25A 
Southern hemisphere 
• KF758847 EC-33A 
• KF758848 G P -C33 
• KF758852 GP-C56 
100 
• KF758856 KZN-C29 
• KF758857 KZN-C31 
• KF758880 NW-B7 
• KF758882 NW-88 
• KF758904 W C-C18 
• KF758905 WC-C20 
• KF758931 LP46 
• KF758932 LP49 
Figure 13b: Neighbor-joining phylogenetic tree of A. marginale msp4 gene sequences from strains 
identified in cattle in South Africa represented with a green dot and blue dot representing an outgroup (A. 
ovis and A phagocytophilum) including isolates from East and North Africa, North and South America, 
Europe and Australia. The phylogenetic tree was implemented in the MEGA5 (Tamura et al., 2011 ). 
Bootstrap analysis was conducted with 1000 replicates. The GenBank accession numbers of the 
respective sequences used for the phylogenetic analysis are indicated at the beginning of the name of 
each sequence. Only bootstrap values higher than 50% are shown. 
82 
5.3.4 The phylogenetic analysis of msp4 gene using maximum-likelihood 
phylogenetic tree 
Observations made from the maximum-likelihood phylogenetic trees in Figure 13a 
further verify the results of the neighbor-joining phylogenetic tree. The only difference 
was that in the second clade the number of bootstrap support significantly decreased. In 
Figure 13b, the difference was noted in the first and second clade when isolates from 
other countries were included with a bootstrap support of 97, respectively. However, the 
South African strains fell into a separate cluster from the rest of the world and other 
African countries. 
83 
Figure 14a: Maximum-likelihood phylogenetic tree of A. marginale msp4 gene sequences from strains 
identified in cattle in South Africa. The phylogenetic tree was implemented in the MEGA5 (Tamura et al. , 
2011 ). Bootstrap analysis was conducted with 1000 replicates. The Gen Bank accession numbers of the 
respective sequences used for the phylogenetic analysis are indicated at the beginning of the name of 
each sequence. 
84 
AF428085 1 Mexico } 
AF428084 1 Mexico 
54 Av2s3100. 1 B razil 4 Southern hemisphere 
AY 191827. 1 Puerto Rico 
6 1 AF428086. 1 Argentina 
AF428083. 1 Mexico 
SB A Y 787172. 1 Israeli round 
JN572928. 1 GZB-China 
AY456002. 1 SP7-Spaln 
AY786994. 1 Israeli tailed 
EU315782. 1 50 (G16)-Hungary 
BB EU315783. 1 51 (G18)-Hungary 
6 • A Y 127076. 1 S tillwater 2 
A Y 010253. 1 Okeechobee 
9 3 A Y 127073. 1 Oklahoma City 
AY010254. 1 Virginia 
63 Northern hemisphere 
A Y010251 . 1 M ississippi 
AY786993 1 lsn1eli non-tailed 
AY702921 . 1 Italy 47 
AY702917. 1 Italy 6 
AF428081 . 1 USA 
77 
AY127065. 1 Oregon 
AY010248. 1 California 
93 AY127077. 1 New Castle 
AY127075. 1 Stillwater 1 
A Y 127068. 1 Glencoe 2 
e AF393742. 1 Anaplasma o...;s 
e AY706389 Anaplasma phagocytophllum 
AY283191 . 1 Brazil 5 
A Y 666007. 1 2 .4-Zlmbabwe 
68 
AY666006. 1 1 .6-Zlmbabwe 
9 3 
A Y666011 . 1 5 .9-Zlmbabwe 
92 AY666004. 1 661Kari-Kenya 
A Y 666003. 1 G38-Australia 
9B AY666002. 1 F72-Australia 
• KF758867 MP-C15 
98 • KF758869 MP-C18 
75 
• KF758845 EC-25A 
• KF758847 EC-33A Southern hemisphere 
• KF758848 GP-C33 
e KF758852 GP-C56 
9B 
• KF758856 KZN-C29 
• KF758857 KZN-C31 
• KF758880 NW-87 
98 • KF758882 NW-88 
• KF758904 WC-C 18 
• KF758905 WC-C20 
• KF758931 LP46 
• KF758932 LP49 
Figure 14b: Maximum-likelihood phylogenetic tree of A.marginale msp4 gene sequences from strains 
identified in cattle in South Africa represented with a green dot and blue dot representing an outgroup 
including isolates from East and North Africa, North and South America, Europe, Asia and Australia. The 
phylogenetic tree was implemented in the MEGA5 (Tamura et al., 2011 ). Bootstrap analysis was 
conducted with 1000 replicates. The GenBank accession numbers of the respective sequences used for 
the phylogenetic analysis are indicated at the beginning of the name of each sequence. Only bootstrap 
values hiqher than 50% are shown. 
85 
5.4 Discussion 
The use of molecular data for inferring phylogenetic trees has now gained considerable 
interest among biologists of different disciplines, and is often used in addition to 
morphological data to study relationships in further detail. The number of repeats found 
in msp 1a  has been used to characterize geographic isolates of A. marginale (Allred et 
al. , 1990; de la Fuente et al., 2002a). In a previous study of A. marginale isolates from 
the United States, de la Fuente and colleagues (2003a) concluded that msp1a is not a 
good marker for the characterization of A. marginale geographic isolates and suggested 
that the genetic heterogeneity observed among A. marginale isolates of Oklahoma 
could be better characterized by use of msp4 gene and protein sequences. 
Furthermore, concurrent analysis of msp1a and msp4 gene sequences provided 
phylogeographic information (de la Fuente et al., 2003a). On a broader geographic 
scale, analysis of New World isolates demonstrated that msp4 sequences, but not 
msp1a DNA or protein sequences, provide phylogeographic information (de la Fuente et 
al., 2007a). These results, together with the finding that multiple msp1a genotypes 
circulate in some geographic regions (e.g., an area of endemicity in Oregon; (Palmer et 
al. , 2001 ), questioned the use of msp1a sequences for identification of geographic 
isolates of A. marginale and suggested that msp 1a  sequences may evolve more rapidly 
(de la Fuente et al., 2007a). 
Overall , these results suggested that msp1 a genotypes most likely reflect the history of 
cattle movement rather than the geographic distribution of A. marginale isolates. Recent 
results have shown that multiple A. marginale genotypes were maintained within a herd 
in an area of endemicity by independent transmission events and that infection with 
more than one genotype per host may have been prevented by a mechanism of 
infection exclusion (Palmer et al., 2001 ; de la Fuente et al. , 2007a; Pohl et al., 2013; 
Cabezas-Cruz et al. , 2013; Mutshembele et al., 2014). 
86 
Therefore, if cattle persistently infected with a new A. marginale genotype were 
introduced to a herd, these genotypes could become establ ished by mechanical and/or 
biological transmission to susceptible cattle. In regions with few introductions of A. 
marginale isolates such as Australia, genotypic variation was found to be minimal (Bock 
and de Vos, 2001 ; Lew et al., 2002). In regions like Oklahoma, where the movement of 
cattle has been extensive, a highly heterogeneous population of A. marginale isolates 
would be expected (de la Fuente et al., 2007a). 
In summary, as demonstrated in previous studies (de la Fuente et al., 2005c), the msp4 
sequences of A. marginale provided sufficient variation to provide phylogeographic 
relationships of A. marginale isolates on a geographic scales. In contrast to the resu lts 
obtained with msp4, DNA and deduced amino acid sequences of msp1a failed to 
resolve phylogeographic patterns of A. marginale isolates and suggested that cattle 
movement and the maintenance of different genotypes by independent transmission 
events explain the genetic heterogeneity observed among isolates of A. marginale in 
Oklahoma and, possibly, in other regions of endemicity (de la Fuente et al. , 2007a). 
5.4.1 Phylogenetic analysis of A. marginale using msp1a gene 
The tandem repeat region of msp 1a  is useful as a genetic marker for analysis of A. 
marginale evolutionary and diversity in endemic areas (de la Fuente et al., 2007a). As 
shown previously, A. marginale is endemic in South Africa (de Waal, 2000). This study 
used a sensitive and specific msp 1a  PCR primer pair (Lew et al., 2002) to study the 
epidemiology of A. marginale in 8 different provinces of South Africa as well as the 
structural analysis of msp1a. As was found in previous studies (Pohl et al., 2013; 
Cabezas-Cruz et al. , 2013; Mutshembele et al., 2014) the msp1a sequences of A. 
marginale isolates from South Africa contained a variable number of tandem repeats in 
the amino-terminal region of the protein, while the remainder of the protein was highly 
conserved. 
87 
Newly described tandem repeats from South African isolates were reported for first time 
in this study (Figure 6) suggesting strains that are unique to this geographical region. 
The presence of different msp1 a genotypes identified in different regions (Cabezas-
Cruz et al., 2013) suggest that msp 1a  sequences, although conserved during 
multiplication of the parasite in the bovine host and after transmission by ticks (Palmer 
et al., 2001 ; Bowie et al. , 2002) are very variable and may contribute to the definition of 
genotype variations within cattle. This heterogeneity among msp 1a  gene sequences 
could be explained by the tandem repeats within a region of high mutability, which 
would be supported by the frequency of variable amino acid positions within 
geographical strain that are higher in this region than in the rest of the protein (Bowie et 
al., 2002). 
Nonetheless analysis of this diversity should take into account cattle movement and the 
consecutive multiple introductions of A. marginale strains in a given region (de la Fuente 
et al., 2010) which could be a non-evolutive mechanism that contributes to the local 
genetic variability. Despite the fact that no geographic phylogenetic relationship was 
found using A. marginale msp1a, the percent of identity among msp1a tandem repeats 
in two different regions differed in this study, which suggest that different genetic 
mechanisms may have been involved in the msp1a amino acid variability. 
Using this phylogenetic pattern South African A. marginale strains EC22 and NW-C2 
(27/13/4/4/37) were likely introduced in KwaZulu-Natal from Eastern Cape or North 
West provinces. This supports the evidence that the strains do not fall in the 
phylogenetic cluster formed by the other sequences from KwaZulu-Natal and notably, 
these strains contained tandem repeat number 4 with primarily 11-strand as secondary 
structure. Recently, the presence of 11- strand in msp1a tandem repeats was found to be 
phylogenetically correlated with non-tick transmission phenotype of A. marginale 
(Cabezas-Cruz et al., 2013). 
88 
This is in contrast with the predominance of a-helices in South African strains that 
correlate with tick transmission phenotype of A. marginale (Cabezas-Cruz et al., 2013). 
Several tick species have been incriminated in A. marginale transmission in South 
Africa (Marufu , 2014) with mechanical and transplacental transmission being reported 
(Aubry and Geale, 2011 ), which may likely also contribute to transmission of South 
African EC22-(27/13/4/4/37) strain. 
Anaplasmosis is widespread in KwaZulu-Natal province (du Plessis et al., 1994) and 
some of the tandem repeats found in our study has also been reported in other regions 
of the world (Videtto et al., 2006; de la Fuente et al. , 2007a; Mtshali et al., 2007). 
Although tandem repeats including 25, 31 , 42 and 43 are common in A. marginale 
isolates in KwaZulu-Natal , a higher proportion of new South African tandem repeats 
were found in this study. This data suggested that conditions in this region promoted 
high genetic variability of A. marginale, a finding in agreement with the discovery that 
tandem repeats in this province had the lowest amino acid identity. High infection rates 
by A. marginale-isolates were observed in Mpumalanga, Gauteng and Eastern Cape. 
The high prevalence is attributed to heavy-tick infestation previously reported in the 
areas by Horak et al (1991) , Walker (1991) and Coetzer et al (1994). In particular R. 
decoloratus, R. microplus and R. evertsi evertsi have been reported in these regions 
and some of these species transmit A. marginale. 
The 20 structure of the msp1a tandem repeats in these provinces was predominantly a-
helix, which would be expected in areas heavily infested by these. In contrast, the lack 
of A. marginale in Northern Cape would be expected in areas where conditions and 
intensive tick control programs contributed to low tick populations. Amino acid variability 
in individual positions along the 29 amino acids of all strains suggested that this position 
is the most variable among isolates worldwide (Cabezas-Cruz et al. , 2013). In addition, 
th is position has a high level of negatively-charged amino acids, a region which is 
associated with transmissibility by ticks (Mutshembele et al. , 2014). 
89 
This study has used structural epidemiology to establ ish for the fi rst time A. marginale 
msp1a structural patterns for different geographic regions. Putative secondary structure, 
identity, and phylogenetic analysis of the msp1a tandem repeats were used to infer 
movement of A. marginale strains and regional specific properties of A. marginale 
msp1a. Therefore, the find ings of the present study will form a basis for futu re research 
where the epidemiology of A. marginale can be correlated with the structure of msp1a 
and tick transmissibility. 
5.4.2 Phylogenetic analysis of A. marginale using msp4 gene 
The analysis of msp4 gene, which has provided evolutionary information about 
geographically distinct A. marginale strains (de la Fuente et al., 2007a), was used in this 
study for phylogenetic analysis for isolates infecting cattle from Limpopo, Mpumalanga, 
North West, Gauteng, KwaZulu-Natal, Eastern Cape and Western Cape. Two clades 
were observed , which consisted of the first (LP, NW, GP, KZN and WC) and the second 
(MP and EC) clade isolates. 
Figure 13a represent the neighbor-joining tree of the isolates from MP and EC formed a 
cluster which was isolated from LP, NW, GP, KZN and WC with well supported 
bootstrap values of 99%. However, it shared high genetic simi larities with both clades 
with a high bootstrap support value of 99%. 
A maximum likelihood phylogenetic tree revealed strong bootstrap of 99% for a clade 
containing isolates of A. marginale msp4 gene from LP, NW, GP, KZN and WC. A 
strong bootstrap support of 82% from MP and EC isolates of South Africa (Figure 13b). 
These results were also observed in phylogenetic tree of A. marginale msp4 gene 
sequences from first clade (LP, NW, GP, KZN and WC) and the second clade (MP and 
EC) isolates of South Africa, North Africa, North and South America, Europe, Australia 
as well as Asia. 
90 
These phylogenetic trees were constructed using neighbor-joining and maximum-
likelihood method (Figure 13a and 13b). Sequence comparison of the msp4 gene was 
recognized as one of the most powerful and precise methods for determining the 
phylogenetic relationships of A. marginale (de la Fuente et al., 2007a). 
South African strain had 100% nucleotide identity with isolates from Kenya, Zimbabwe 
and Australian. Even though they had highly shared the identity, South African strains 
remained in separate clusters. These strains shared 99% identity with isolates from 
North and South America, Europe and Asia (Appendix 4). Good representation of 
southern and northern hemisphere strains was observed, which further demonstrated 
that msp4 gene may serve as good marker for phylogeographic analysis (Figure 13a 
and 13b). In addition, these result suggest that South African cattle could be be infected 
with the same strains that infect cattle from West, East and North Africa, North and 
South America, Europe, Asia as well as Australia. 
The DNA sequences of msp4 gene revealed phylogeographical segregation with South 
African isolates and other isolates submitted in the NCBI database. MSP4 gene 
sequences provided phylogenetic resolution for intraspecific relationships among 
isolates. South African isolates demonstrated apparent phylogeograph ic information. 
The present study revealed that msp4 nucleotide sequences are sufficiently variable 
and like in other studies could be used to detect phylogeographic patterns broadly (de la 
Fuente et al., 2010). Therefore, the msp4 gene may be used as a marker for 
understanding the phylogeographic patterns and phylogenetic relationships of A. 
marginale in South Africa. 
91 
CHAPTER 6 
GENERAL DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS 
6.1 General discussion 
The prevalence of bovine anaplasmosis in the various South African regions is often 
complex and may involve the bovine population immunity to A. marginale, the means of 
transmission (mechanical, biological by ticks, transplacental or a combination thereof) , 
as well as the genetic variability of A. marginale strains within a given area. In this 
study, data was not collected on the immunity of cattle populations for A. marginale. 
Certainly, tick transmission plays a very important role in the prevalence of A. 
marginale, and the distribution of tick species in South Africa has been determined by 
de Waal, (2000). The provinces with 100% prevalence of A. marginale infected cattle 
did not correlate with provinces with the highest or lowest A. marginale genetic diversity, 
but instead represented a range of diversity (Figure 7). It has been hypothesized that 
increased genetic variability may have an impact on A. marginale strains transmission 
(Palmer and Bryton, 2013). 
Alternately, A. marginale strains with low genetically variability may be lost after immune 
clearance. Interestingly, in the Western Cape the prevalence of A. margina/e was high 
while the genetic diversity was low, which could be due to low immunity of cattle 
population in the particular sample points. Overall , the prevalence of A. marginale in 
cattle from South Africa is high when compared with other regions of the world. In Brazil 
a recent study found prevalence of A. marginale in a cattle herd to be 70% (Pohl et al., 
2013). Another study by de la Fuente et al. (2005a) reported the prevalence of A. 
marginale in cattle herds from Italy to range from 25 - 100%. 
92 
In these studies the prevalence and genetic diversity of A marginale were found to 
have a polynomial, and nonlinear correlation rather than a linear one. Subsequently, this 
finding most likely reflects the complexity of factors that may be influencing the 
prevalence of A marginale in the various regions of South Africa as evident in the 
province of Limpopo, North West and KwaZulu-Natal. This polynomial correlation 
suggests that increased genetic diversity impacted on the transmissibility of A 
marginale strains. The other major factor that could influence A margina/e prevalence 
in a given region is the immunity of a population . 
This factor is also related to genetic diversity because, as shown previously, a 
mechanism of infection exclusion may prevent cattle infected with one genotype from 
becoming infected with a second closely related one. However, widely separated 
genotypes apparently can be maintained in cattle as co-infections which could 
contribute to wider genetic diversity at the population level and impact on the 
prevalence of bovine anaplasmosis. Anap/asma marginale with low genetic variabil ity 
will likely become highly prevalent in a cattle population with predominately na"ive 
animals, without immunity as evidenced in the cattle population in the Western Cape. 
6.2 Conclusions 
The regional prevalence of bovine anaplasmosis appears to be a result of complex 
factors, including the immunity of cattle populations to A marginale, the mode of 
transmission (biological by ticks, mechanical and/or transplacental) and the genetic 
variability of A. marginale strains within cattle populations. These factors have 
contributed to the challenges of developing effective diagnostic and control measures 
for A. marginale. Molecular tools are required for evaluating the genetic diversity of A. 
marginale. Developing an understanding of the molecular mechanisms underlying the 
generation of genetic diversity is crucial in implementation of effective control measures 
that are developed in concern with evolutionary changes of the pathogen which do not 
stay one step behind in pathogen evolution. 
93 
The first step in this molecular study of bovine anaplasmosis was to confirm that A. 
marginale was widespread in various regions in South Africa. The genetic diversity of A. 
marginale msp1a was then characterized , and found to arise from the evolution of 
extent tandem repeats, which under positive and negative selection, diversified as noted 
by the presence of newly reported tandem repeats variants. These new variants were 
most likely constricted by two forces: (1) the immune system and (2) biological 
transmission of A. marginale by ticks. Common msp1a tandem repeats were found 
among A. marginale strains in South Africa and other regions of the world which , in 
combination with the use of low variable msp 1a  peptides, will likely be useful in the 
development of msp 1a  based vaccine for more effective regional control of 
anaplasmosis. 
Gene sequences of msp4 provided sufficient variation to allow for the characterization 
of phylogeographic patterns on a broad scale. The South African clade of A. marginale 
msp4 gene sequences included isolates from West, East and North Africa, North and 
South America, Europe, Australia and Asia (Appendix 4). Finally the detection of diverse 
clades of A. marginale representing different geographic regions including the results 
that were obtained based on msp1a gene sequences may provide vital information for 
developing new novel vaccine approaches for control of anaplasmosis. 
This work presents information on the prevalence, genetic diversity and phylogenetic 
analysis of A. marginale in cattle throughout South Africa. Data compiled in this study 
will aid in the future management of A. marginale infection and may provide a molecular 
basis for targeted vaccine development. The information gained from this research 
provides an understanding of the epidemiology of A. margina/e in South Africa which 
will be fundamental toward the development of diagnostic assays and control measures 
for bovine anaplasmosis. 
94 
6.3 Recommendations 
6.3.1 To further determine the species, distribution and population densities of the 
known tick vectors of A. marginale in the provinces of South Africa , and to 
correlate these tick populations with the prevalence of bovine anaplasmosis. 
6.3.2 To characterize A. marginale strains in cattle populations in order to determine 
the strain diversity, and to conduct phylogenetic studies of these strains to gain 
insight into the origin and evolution of A. marginale in South Africa. 
6.3.3 To identify the tick species infesting cattle in anaplasmosis endemic regions , and 
to determine the immunity of cattle in these areas for A. marginale infections. 
6.3.4 To evaluate genes and gene products of the A. marginale strains, and to 
determine whether the diversity of msp4 gene is related to selective pressures. 
95 
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131 
APPENDICES 
Appendix1: Raw data of statistical analysis of A marginale in cattle in studied 
provinces of South Africa 
The table below qives the qeneral outcome 
. tab Anapla sma_results nw 
ANA PLASMA I 
MARGINALE I 
PCR RESULTS I Freq . Percent Cum . 
- -----------+----------- ------------- -----------
Negative I 71 25 . 36 
Positive I 209 100 . 00 
- - -- - - - -----+------- - ---- - ----------------------
Total I 280 100 . 00 
. prop Anaplasma_ results_ nw 
Proportion estimation Number of obs 280 
I Pr oportion Std . Err . [95% Conf . Interval) 
--- ------------------+---------------- -------------- ------------------
Anaplasma_ resul ts nw I 
Negative I . 0260461 . 2057817 . 308153 4 
Positive I . 0260461 . 6918 466 . 7942183 
. tab Anaplasma_results nw farm_nw , exact 
ANAPLASMA 
MARGI NALE 
PCR I FARM 
RESULTS I Commer cia Communal I Total 
-----------+----------------------+----------
Negati ve I 4 67 I 71 
Pos it i ve I 61 148 I 209 
---------- -+----------------------+--- - - -----
Total I 65 215 I 280 
Fi sher ' s exact 
1-si ded Fisher ' s exact 0 . 000 
132 
. prop Anaplasma_results_nw , over( farm_nw) 
Proportion estimation Number of obs 280 
Negative: Anaplasma_resul t s_nw Negative 
Positive : Anaplasma_results_nw Positive 
Commercial : farm nw =Commercial 
Communal : farm nw = Communal 
Over I Proportion Std . Err. [ 95% Conf. Interva l) 
-------------+------------------------------------------------
Negative 
Commercial I . 0615385 . 030039 4 . 0230112 . 1543787 
Communa l I . 3116279 . 0316609 . 2529283 . 3770745 
-------------+--------------------------------------------- ---
Positive 
Commercial I . 0300394 . 84 56213 . 9769888 
Communal I . 0316609 . 6229255 . 7470717 
. kwallis Anaplasma_results_nw , by( location_nw) 
Kruskal-Wallis equality-o f-populations rank test 
+--------------------------------+ 
location_nw I Obs I Rank Sum I 
!---------------+-----+----------! 
I Eastern Cape I 40 I 7040 . 00 I 
I Gauteng I 20 I 3520 . 00 I 
I KwaZulu -Natal I 55 I 8700 . 00 
I Limpopo I 40 I 5920 . 00 I 
I Mpumalanga I 21 I 3696 . 00 I 
!---------------+-----+----------! 
I North West I 33 I 4828 . 00 I 
I Northern Cape I 45 I 1620 . 00 I 
I Western Cape I 26 I 4016 . 00 I 
+------------------- -------------+ 
chi- squared 94 . 425 with 7 d . f . 
probability 0 . 000 1 
133 
The hypothesis that the farms are similar with respect to prevalence is 
rejected by the results above 
. tab location_nw Anaplasma_results_nw , row col cell chi2 
+-------------------+ 
I Key I 
1-------------------1 
I frequency I 
I row percentage 
I column percentage 
I cell percentage I 
+-------------------+ 
I ANAPLASMA MARGINALE 
I PCR RESULTS 
LOCATION I Negative Positive Total 
--------------+----------------------+----------
Eastern Cape I O 40 I 40 
o. oo 100 . 00 I 100 . 00 
0 . 00 I 14 . 29 
0 . 00 I 14.29 111111 
--------------+----------------------+----------
Gauteng I 0 20 I 20 
I 0 . 00 100 . 00 I 100 . 00 
I 0 . 00 .. I 7 . 14 
I o. oo 7 . 14 I 7 . 14 
--------------+----------------------+----------
KwaZulu-Natal I 7 48 I 55 
I 12 . 73 87 . 27 I 100 . 00 
I 9 . 86 I 19 . 64 
I 2 . 50 I 19 . 64 
--------------+--- -------------------+----------
Limpopo I 8 32 I 40 
I 20 . 00 80 . 00 I 100 . 00 
I 11 . 27 111111 I 14 . 29 
I 2 . 86 11 . 43 I 14 . 29 
--------------+--- -------------------+----------
Mpumalanga I 0 21 I 21 
I 0 . 00 100 . 00 I 100 . 00 
I 0 . 00 I 7 . 50 
I 0 . 00 I 7 . 50 
--------------+----------------------+----------
North West I 7 26 I 33 
I 21. 21 78 . 79 I 100 . 00 
I 9 . 86 111111 I 11 . 79 
I 2 . 50 9 . 29 I 11 . 79 
--------------+----------------------+----------
Northern Cape I 4 5 0 I 4 5 
I 100 . 00 o. oo I 100 . 00 
I 63 . 38 o. oo I 16 . 07 
I 16 . 07 .. I 16 . 07 Lowest (Northern Cape) 
--------------+----------------------+----------
134 
Western Cape I 4 22 I 26 
15 . 38 8 4 . 62 I 100 . 00 
5 . 63 - I 9 . 29 
1. 43 7 . 86 I 9.29 
--------------+----------------------+------ ----
Total I 71 209 I 280 
I 25 . 36 74 . 64 I 100 . 00 
I 1 00 . 00 100 . 00 I 100 . 00 
I 25 . 36 74 . 64 I 100.00 
Pearson chi2(7) = 1 66 . 8890 Pr 0 . 000 
. prop Anaplasma results nw if farm nw==lllllllllllll 
Proport i on estimation Number of obs 65 
I Proportion Std . Er r . [95% Cont . Interval] 
-- - - --------- -- ------+-- --- ------------------------------- ------------
Anaplasma_ results_ nw I 
Negative I . 0615385 . 0300394 . 0226719 . 1563737 
Positive I . 938461 5 . 0300394 . 843 6263 . 977 32 81 
~aplasm 
Proportion estimation Number o f obs 215 
I Pr oportion St d . Err. [95% Conf. Inte r val ] 
- - --------------- ----+----------------- ----------------------------- - -
Anaplasma_ result s nw I 
Negat i v e I . 3116279 . 0316609 .2528555 . 377 165 
Positive I . 6883721 . 0316609 . 622835 . 7471445 
tab farm nw 
FARM I Freq . Percent Cum . 
----- -------+-----------------------------------
Commercial I 65 23 . 2 1 23 . 21 
Communal I 215 76 . 79 100 . 00 
----- - ------+----- ------------ ------------ - -----
Total I 280 100 . 00 
. kwallis location_ nw if Anaplasma_results_nw==O , by( farm nw) 
Kruskal-Wallis equality-of-populations rank test 
+------------- ------ - ---------+ 
I farm nw I Obs I Rank Sum I 
135 
1------------+- -- - -+----------1 
I Commercial I 4 I 27 8 . 00 I 
I Communal I 67 I 2 278 . 00 I 
+-- - ----- - - -------- - - - - -------+ 
c hi-squared 11 . 167 wi th 1 d . f . 
p r obability 0 . 000 8 
chi - squared with ties = 15 . 049 wi t h 1 d . f . 
probabil i t y 0 . 000 1 
. kwalli s loc ation_nw i f Anaplasma_ r esults_lllllllllll) , by( farm nw) 
Kruskal - Wall is equa lity- o f - population s rank t es t 
+----- - -- - - - - --- - -------------+ 
farm_nw I Obs I Ran k Sum I 
!--- - - ------- +--- - -+- ------- - - ! 
I Commerc ial I 61 I 5 7 66 . 50 I 
I Communal I 148 I 1 61 78 . 50 I 
+--------------- - --- - ---- ---- -+ 
chi- square d 2 . 580 with 1 d . f . 
p r obabi l i t y 0 . 1082 
c h i-squared with t ies = 2 . 654 with 1 d . f . 
prob a bility 0 . 1033 
. tab Anaplasma_results nw 
ANAPLASMA I 
MARGINALE I 
PCR RESULTS I Fr e q . Per c ent Cum. 
- - ----------+-- - - --- ---------- - -- - ----------- -- -
Negativ e I 71 2 5 . 3 6 2 5 . 3 6 
Po s itive I 209 74. 64 1 0 0 . 00 
- - - - -- - - - ---+- - --- --- - ---- - - -- - ----- - ------ -----
To tal I 280 100 . 00 
. p r op location_ nw if Anaplasma_results_nw== l~) , ove r ( f a rm nw) 
Propo rtio n estimatio n Number o f obs 209 
Over I Prop o rtio n Std . Err . [ 95% Conf. Interv al ) 
- ------ - - ----+- ----- - --- --- - ------ - -- - ----------- - - - ---- ------
Eastern Cape I 
Commerc i al I . 31147 5 4 . 05978 55 . 2070 474 . 4393882 
136 
Communal I .14 18919 . 02878 . 0940079 . 2085517 
-------------+------------------------------------------------
Gauteng 
Commercial I . 3278689 . 060604 . 2209735 . 4561933 
Communal I . (no observations) 
-------------+------------------------------------------------
KwaZulu-Natal I 
Commercial I (no observations) 
Communal I . 3243243 . 03861 . 2532568 .4 0 4531 
-------------+------------------------------------------------
Limpopo 
Commercial I (no observat ions ) 
Communal I . 2162162 . 0339534 . 1567198 . 2905182 
-------------+------------------------------------------------
Mpumalanga I 
Commercial I (no obse r vatio ns) 
Communal I .14 18919 . 02878 . 09 40079 . 2085517 
-------------+------------------------------------------------
North West I 
Commercial I (no observations) 
Communal I .1 756757 . 0313867 . 1220444 . 2 462639 
-------------+------------------------------------------------
Western Cape I 
Commercial I . 3606557 . 061992 4 . 2 4 92643 .4 89377 
Communal I . (no observ ations) 
. prop location_nw if Anaplasma_results nw==l & farm nw==l , over( 
farm_nw) 
Proportion estimation Number of obs 61 
Commercial : farm nw -
Over I Proportion Std. Err . (95 % Conf . Interval) 
-------------+-------------------------- ----------------------
Eastern Cape I 
Commercial I .3114 7 54 . 0597855 . 2057294 . 4413712 
-------------+-------------------------- ----------------------
Gauteng I 
Commerc ial I • 3278689 . 06060 4 . 2 1961 01 . 4581632 
-- -----------+----------- -------------------------------------
Western Cape I 
Commercial I . 3606557 . 0619924 . 24 7815 . 4913163 
. prop l ocation_nw if Anaplasma_results_ nw==l & , over( 
farm_nw) 
Over I Prop ortion Std . Err . (95% Conf. I nterval) 
137 
-------------+------------------------------------------------
Eastern Cape I 
Communal I . 1418919 . 02878 . 0939113 . 208739 
-------------+------------------------------------------------
KwaZulu - Natal 1 
Communal I . 3243243 . 03861 . 253097 . 4047346 
-------------+------------------------------------------------
Limpopo 
Communal I . 2162162 . 0339534 . 1565927 . 2907164 
-------------+------------------------------------------------
Mpumalanga 
Communal I . 1418919 . 02878 . 0939113 . 2087 39 
-------------+------------------------------------------------
North West I 
Communal I . 1756757 . 0313867 . 121933 . 2464571 
. prop location_nw if Anaplasma results_nw==O , over ( farm nw) 
Proporlion estimation Number of obs 71 
Commercial : farm nw Commercial 
Communal : farm nw =Communal 
Over I Proportion Std . Err . [95% Conf . Interva l) 
-------------+------------------------------------------------
KwaZulu - Natal I 
Commercial I . (no observations ) 
Communal I . 1044776 . 0376511 . 0496877 . 206552 
-------------+------------------------------------------------
Limpopo 
Commercial I . (no observations) 
Communal I . 119403 . 0399139 . 059794 . 2242624 
-------------+------------------------------------------------
North West I 
Commercial I . (no observat i ons) 
Communa l I . 10 4 4776 . 0376511 . 0496877 . 206552 
-------------+--------- - --------------------------------------
Northern Capel 
Commercial I . (no observations) 
Communal I . 6716418 . 0578057 . 5480652 . 7752826 
-------------+-------------- - ---------------------------------
Western Cape I 
Commercial I 1 0 
Communal I . (no observations) 
138 
Appendix 2: GenBank accession number of Anaplasma marginale msp1a gene 
isolates and their origin in South Africa 
Sample names Origin Gen Bank accession 
numbers 
LP-37 Limpopo (KC470153) 
LP-46 Limpopo (KC470154) 
LP-7 Limpopo (KC470155) 
LP-10 Limpopo (KC470156) 
LP-50 Limpopo (KC470157) 
LP-30 Limpopo (KC470158) 
LP-34 Limpopo (KC470159) 
MP-C2 Mpumalanga (KC4701 60 
MP-C5 Mpumalanga (KC470161) 
NW-C2 North West (KC4701 62) 
NW-C4 North West (KC470163) 
NW-C5 North West (KC4701 64) 
NW-C1-160312 North West (KC470165) 
NW-C4-160312 North West (KC470166) 
GP-C1 Gauteng (KC470167) 
GP-C2 Gauteng (KC470168) 
GP-C5 Gauteng (KC470169) 
139 
GP-C1112105 Gauteng (KC470170) 
GP-C4117105 Gauteng (KC470171 ) 
GP-C1817105 Gauteng (KC470172) 
GP-C7117105 Gauteng (KC470173) 
KZN-F KwaZulu-Natal (KC470174) 
KZN-MN KwaZulu-Natal (KC470175) 
KZN-K KwaZulu-Natal (KC470176) 
KZN-D KwaZulu-Natal (KC470177) 
KZN-Y KwaZulu-Natal (KC470178) 
KZN-14 KwaZulu-Natal (KC470179) 
KZN-19 KwaZulu-Natal (KC470180) 
KZN-49 KwaZulu-Natal (KC470181 ) 
KZN-51 KwaZulu-Natal (KC470182) 
EC-22 Eastern Cape (KC470183) 
EC-23 Eastern Cape (KC470184) 
EC-24 Eastern Cape (KC470185) 
WC-4 Western Cape (KC470186) 
WC-6 Western Cape (KC470187) 
WC-7 Western Cape (KC470188) 
WC-8 Western Cape (KC470189) 
WC-10 Western Cape (KC470190) 
140 
WC-11 Western Cape (KC470191) 
WC-12 Western Cape (KC470192) 
WC-13 Western Cape (KC470193) 
WC-14 Western Cape (KC470194) 
WC-15 Western Cape (KC470195) 
WC-16 Western Cape (KC470196) 
141 
Appendix 3: GenBank accession number of Anap/asma marginale msp4 gene 
isolates and their origin in South Africa 
Samples name Origin Gen Bank accession 
number 
LP-C1 Limpopo Province KF758907 
LP-C2 Limpopo Province KF758909 
LP-C3 Limpopo Province KF758910 
LP-C7 Limpopo Province KF758911 
LP-C9 Limpopo Province KF758912 
LP-C10 Limpopo Province KF758913 
LP-C11 Limpopo Province KF758914 
LP-C14 Limpopo Province KF758915 
LP-C19 Limpopo Province KF758916 
LP-C21 Limpopo Province KF758917 
LP-C23 Limpopo Province KF758918 
LP-C27 Limpopo Province KF758919 
LP-C29 Limpopo Province KF758920 
LP-C30 Limpopo Province KF758921 
LP-C32 Limpopo Province KF758922 
LP-C33 Limpopo Province KF758923 
LP-C34 Limpopo Province KF758924 
142 
LP-C35 Limpopo Province KF758925 
LP-C38 Limpopo Province KF758926 
LP-C40 Limpopo Province KF758927 
LP-C41 Limpopo Province KF758928 
LP-C43 Limpopo Province KF758929 
LP-C46 Limpopo Province KF758930 
LP-C49 Limpopo Province KF758931 
LP-C50 Limpopo Province KF758932 
LP-C77 Limpopo Province KF758933 
LP-C85 Limpopo Province KF758934 
MP-C1 Mpumalanga Province KF758860 
MP-CS Mpumalanga Province KF758862 
MP-C? Mpumalanga Province KF758864 
MP-C8 Mpumalanga Province KF758866 
MP-C15 Mpumalanga Province KF758867 
MP-C18 Mpumalanga Province KF758869 
MP-C19 Mpumalanga Province KF758871 
MP-C20 Mpumalanga Province KF758872 
MP-C21 Mpumalanga Province KF758874 
NW-CB3 North West Province KF758842 
NW-CB4 North West Province KF758876 
143 
NW-CBS North West Province KF7S8878 
NW-CB? North West Province KF7S8880 
NW-CB8 North West Province KF7S8882 
NW-C28 North West Province KF7S8883 
GP-C33 Gauteng Province KF7S8847 
GP-C48 Gauteng Province KF7S8849 
GP-CS6 Gauteng Province KF7S88S2 
KZN-C26 KwaZulu-Natal Province KF7S88S4 
KZN-C29 KwaZulu-Natal Province KF7S88S6 
KZN-C31 KwaZulu-Natal Province KF7S88S7 
KZN-C37 KwaZulu-Natal Province KF7S88S8 
KZN-C42 KwaZulu-Natal Province KF7S88S9 
EC-C6A Eastern Cape Province KF7S8843 
EC-C2SA Eastern Cape Province KF7S884S 
EC-C33A Eastern Cape Province KF7S8847 
WC-C4 Western Cape Province KF7S888S 
WC-CS Western Cape Province KF7S8886 
WC-C6 Western Cape Province KF7S8888 
WC-C7 Western Cape Province KF7S8890 
WC-C8 Western Cape Province KF7S8892 
WC-C9 Western Cape Province KF7S8894 
144 
WC-C10 Western Cape Province KF758896 
WC-C12 Western Cape Province KF758898 
WC-C15 Western Cape Province KF758900 
WC-C16 Western Cape Province KF758902 
WC-C18 Western Cape Province KF758904 
WC-C20 Western Cape Province KF758905 
145 
r 
I 
f 
Appendix 4: Amplified sequences of Anaplasma spp. isolates , their origin and 
GenBank accession number 
Isolates and their origin GenBank accession number 
California AY010248.1 
GZ8-China JN572928.1 
51 (G18)-Hungary EU315783.1 
New Castle AY127077.1 
Glencoe 2 AY127068.1 
Stillwater 1 AY127075.1 
AY851150.1 Switzerland 
Oregon AY127065.1 
661 Kari-Kenya AY666004.1 
USA AF428081 .1 
1.6-Zimbabwe AY666006.1 
2.4-Zimbabwe AY666007.1 
5.9-Zimbabwe AY666011 .1 
Italy 6 AY702917.1 
Italy 47 AY702921 .1 
Israeli non-tailed AY786993.1 
50 (G16)-Hungary EU315782.1 
Mississippi AY010251 .1 
146 
Virg inia AY010254.1 
Oklahoma City AY127073.1 
Israeli tailed AY786994.1 
Okeechobee AY010253.1 
Stillwater 2 AY127076.1 
Israeli round AY787172.1 
SP?-Spain AY456002.1 
TWN1-Taiwan EU677383.1 
Mexico AF428083.1 
Mexico AF428084.1 
Mexico AF428085.1 
Brazil 4 AY283190.1 
Brazil 5 AY283191 .1 
Puerto Rico AY191827.1 
F72-Austral ia AY666002.1 
G38-Austra lia AY666003.1 
Argentina AF428086.1 
Anaplasma phagocytophilum AY706389 
Anaplasma ovis AF393742.1 
147