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Molecular characterization of live

Theileria parva sporozoite vaccine
stabilates reveals extensive genotypic
diversity

Article in Veterinary Parasitology · February 2011

Impact Factor: 2.46 · DOI: 10.1016/j.vetpar.2011.01.057 · Source: PubMed

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 Veterinary Parasitology 179 (2011) 62–68

Contents lists available at ScienceDirect

Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Molecular characterization of live Theileria parva sporozoite vaccine

stabilates reveals extensive genotypic diversity
Ekta H. Patel a,∗ , Donald M. Lubembe b , James Gachanja a , Stephen Mwaura a , Paul Spooner a ,
Philip Toye a
a
International Livestock Research Institute, P.O. Box 30709-00100, Nairobi, Kenya
b
University of Nairobi, P.O. Box 30197 G.P.O., Nairobi, Kenya

a r t i c l e i n f o a b s t r a c t

Article history: The current Infection and Treatment Method of vaccination against East Coast fever com-
Received 22 November 2010 prises an inoculation of live Theileria parva sporozoites and simultaneous administration
Received in revised form 20 January 2011
of oxytetracycline. Immunization with a combination of parasite types has been shown to
Accepted 27 January 2011
provide broader protection than inoculation of individual strains. In this study, we used
a high-throughput capillary electrophoresis system to determine the genotypic compo-
Keywords:
sition of the Muguga Cocktail, a widely used vaccine stabilate derived from three seed
Theileria parva
stabilates—Muguga, Serengeti-transformed and Kiambu 5. Five satellite markers were used
East Coast fever
Infection and Treatment Method to genotype the vaccine and reference stabilates from two commercial-scale preparations
Vaccine of the vaccine. In addition, 224 cloned cell lines established by infection of bovine lympho-
cytes with T. parva parasites from the component stabilates were genotyped. The results
indicate that, for the recently prepared batch, there are at least eight genotypes in each
of the Muguga and the Serengeti-transformed stabilates, while parasites from the Kiambu
5 stabilate showed no diversity at the five loci. The Serengeti-transformed stabilate con-
tained parasites of the Kiambu 5 genotype and of two genotypes present in the Muguga
stabilate, whereas there were no genotypes common to the Muguga and Kiambu 5 stabi-
lates. When stabilates from the two vaccine batches were compared, no allelic variations
were identified between the Muguga and Kiambu 5 parasites, while lack of sufficient clones
prevented a full comparison of the Serengeti-transformed stabilates. The findings will facil-
itate examination of the extent to which the vaccine strains become resident in areas under
vaccination, the identification of ‘breakthrough’ strains and the establishment of the qual-
ity assurance protocols to detect variations in the production of the vaccine. The cloned
cell lines will be useful for further understanding the antigenic diversity of parasites in the
vaccine.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction a further 28 million of the 47 million cattle in the region

East Coast fever (ECF) is a fatal bovine disease caused by
the intracellular protozoan parasite Theileria parva. In the
11 countries in central, eastern and southern Africa where
the disease is found, about 1 million cattle per year die, with
∗ Corresponding author. Tel.: +254 20 4223000x4604.
E-mail address: e.patel@cgiar.org (E.H. Patel).
being at risk of contracting the disease. The economic losses
are estimated to be greater than US$300 million per year
(Ndegwa, 2005). The parasite is transmitted by the brown
ear tick Rhipicephalus appendiculatus and models for pre-
dicting the impact of climate change on tick-borne disease
and tick distribution suggests that the number of cattle
at risk of ECF will increase (Olwoch et al., 2008). Infec-
tion of susceptible cattle results in a rapid proliferation of
infected lymphocytes and death within two to three weeks

0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2011.01.057
 E.H. Patel et al. / Veterinary Parasitology 179 (2011) 62–68
(Irvin and Mwamachi, 1983). Animals that survive infec-
tion, either naturally or following treatment, appear to be
solidly immune to further disease. The immunity is thought
to be mediated by killing of infected lymphocytes by CD8+
T cells (McKeever et al., 1994). There is evidence for strain
specificity in the immune response, such that some cat-
tle immunized with one stock are susceptible to challenge
with heterologous stocks. Although the molecular basis of
the strain specificity is not clearly established, there is evi-
dence suggesting that it may be due to diversity in the
antigens presented by the class I MHC molecules on the
surface of infected cells (MacHugh et al., 2009).
The observation of naturally acquired immunity in
infected animals has led to the development of a vaccina-
tion method using live sporozoites (Radley et al., 1975).
The vaccination regime, known as the Infection and Treat-
ment Method (ITM), comprises inoculation of cattle with
a homogenate of infected ticks simultaneously with a sin-
gle dose of a long-acting formulation of oxytetracycline to
curtail the infection. It appears to be important to include
several T. parva stocks in the vaccine to induce a broadly
protective immunity (Radley et al., 1975), at least in east-
ern Africa where there is considerable heterogeneity in
the parasite populations (Odongo et al., 2006). The most
widely used ITM vaccine stabilate in this region is the
‘Muguga Cocktail’, which includes parasites from three
stocks: Muguga, Serengeti-transformed and Kiambu 5. The
Muguga Cocktail is the focus of a multi-institute effort
to commercialize the production and distribution of this
method of control of ECF (GALVmed, 2010).
Many scientists have acknowledged that the
widespread deployment of the ITM vaccine has been
hindered by several concerns. First, the vaccination pro-
cess induces a carrier state in immunized animals, which
may lead to the introduction and eventual dominance of
parasite types derived from the vaccine into areas previ-
ously free of these types (Oura et al., 2007). Second, there
is the possibility of ‘breakthroughs’ from parasite strains
antigenically distinct from the vaccine stabilates, which
may require the incorporation of additional stocks into the
vaccine. The identification of such breakthrough strains
is essential to understanding and addressing this threat.
Third, the commercial-scale production of the vaccine
requires that each batch of the vaccine is antigenically
consistent and that antigenically important parasites are
not lost from the vaccine seed stabilates during passage
through ticks and cattle.
A central issue in addressing these constraints to the
ITM vaccine is a deeper understanding of the composi-
tion of the Muguga Cocktail stabilate. Earlier studies using
monoclonal antibody profiles, repetitive DNA probes, PCR
analysis of antigen genes and mini- and microsatellite
markers indicated that the three seed stabilates con-
sist of at least six parasite types and that the Muguga
and Serengeti-transformed stabilates are very similar
(Oura et al., 2004, 2007). The latter observation ques-
tions the need to include both of these stabilates in
the vaccine. The results were obtained on uncloned cell
lines and contrast with those of Katzer et al. (2006),
who discovered 48 parasite types in a population of
cloned cell lines derived from another T. parva stabilate.
63
The emergence of a dominant parasite type within the
stabilate was apparent as over 75% of the clones con-
tained parasites of the same genotype. These observations
have obvious implications for the consistent production
of ITM vaccines. The present studies were undertaken
to obtain a greater understanding of the composition
of the Muguga Cocktail through satellite genotyping of
vaccine stabilates and cloned cell lines derived from
them.
2. Materials and methods
2.1. Parasite material
The ITM vaccine stabilates FAO 1 and ILRI 0804 were
produced in 1996 and 2008, respectively, from cattle indi-
vidually infected with one of the seed stabilates Muguga
73, Serengeti-transformed 69 or Kiambu 5 68. Ticks were
removed from the cattle, pooled and homogenized to pro-
duce the vaccine stabilates (Radley et al., 1975). In addition
to the vaccine stabilates, individual reference stabilates
were produced from similar batches of ticks derived from
the same cattle infected with the respective seed stabi-
lates. The reference stabilates were named Muguga 4138,
Serengeti-transformed 4139, and Kiambu 5 4137 for FAO
1, and Muguga 4230, Serengeti-transformed 4229, Kiambu
5 4228 for ILRI 08. DNA was also prepared from the blood
of an animal infected with the Muguga cloned sporozoite
stabilate 3308 (Morzaria et al., 1995) to determine the
expected allele sizes for T. parva Muguga using the five
satellite markers.
DNA was extracted using a commercial kit according
to the manufacturer’s instructions (Qiagen DNeasy® Blood
and Tissue Kit).
2.2. In vitro infection and cloning
In vitro infection and cloning of infected lymphocytes
was undertaken according to the method of Goddeeris
and Morrison (1988). Briefly, peripheral blood mononu-
clear cells (PBMC) were isolated from defibrinated jugular
venous blood by flotation on Ficoll-Paque (Pharmacia). The
PBMC (107 ) were resuspended in 1 ml of culture medium
and mixed with an equal volume of sporozoite supernatant.
The supernatant was prepared by centrifuging thawed vac-
cine stabilate at 4200 rpm for 2 min to remove tick debris.
The mixture was incubated for 2 h at 37 ◦ C with occasional
agitation. Additional RPMI 1640 medium was added to
the mixture, followed by centrifugation 200 × g for 10 min.
Infected cells were resuspended in culture medium at a
density of 2.5 × 106 cells/ml and dispensed in a 24-well
plate in 2 ml culture medium per well. The plate was incu-
bated for 48 h at 37 ◦ C in a humidified atmosphere of 5%
CO2 in air. Cells were harvested, assessed for viability by
trypan blue exclusion and suspended in culture medium
at a density of 105 viable cells/ml. This suspension was
used to seed 96-well round-bottom plates as per follows:
1 × 104 /well, 3 × 103 /well, 1 × 103 /well, 3 × 102 /well and
1 × 102 /well in aliquots of 100 ␮l/well. Each well received
5 × 104 irradiated (50 Gy) PBMC filler cells derived from the
same animal in 100 ␮l of cultured medium supplemented
64 E.H. Patel et al. / Veterinary Parasitology 179 (2011) 62–68
Table 1
Minisatellite (MS) and microsatellite (ms) markers used in this study, together with the sizes of the amplicon predicted from the genome sequence and
that observed by capillary genotyping of DNA from the F100 Tp Muguga cloned cell line.
Satellite markersa PCR primer pairs and fluorescent labels
ms 9 (3) VIC® -ctg gtt cct cat ctt cac act a
ctt tcc aga acc tac aat cac
MS 7 (1) NEDTM -gtt cag tcc tat ggc aat tca g
caa acc tct tca aat tca ctc tag g
MS 19 (2) PET® -cca gac acc tca aat ccc aag ta
cca cac tgc cac cta ata caa a
MS 25 (3) VIC® -aca cac cca tca acg tag taa c
cac cat cac act ctt aac cat
MS 39 (4) NEDTM -cca atc aac atc aac tac tcc
cga act cca aac gat cta aac
a
Numbers in brackets refer to the chromosome in which the marker is located.
with 50% conditioned medium derived from established
T. parva-infected lymphoblast cultures. Plates were incu-
bated at 37 ◦ C in a humidified atmosphere of 5% CO2 in
air for 2–3 weeks and screened for the presence of sin-
gle clones. For genotyping analysis, DNA was extracted
using a commercial kit (Qiagen DNeasy® Blood and Tis-
sue Kit) using the manufacturer’s instructions. In some
instances, the PCR was performed directly on cloned cells
(1–2 ␮l from the wells) after denaturation in mineral
oil, essentially as described in Hobson-Peters and Toye
(2005).
2.3. Polymorphic markers and genotyping
The satellite markers used in this study have been pre-
viously described by Oura et al. (2003) and are listed in
Table 1, together with the sequences of the PCR primers.
The markers were selected on the basis of being highly
polymorphic and with at least one marker on each of the
four chromosomes. One primer of each pair was labelled
at the 5 end with one of the following fluorophores:
VIC® green dye label, NEDTM yellow dye label or PET® red
dye label (Applied Biosystems).
2.4. Touchdown (TD) PCR amplification of the mini- and
microsatellite loci
Amplifications were performed using whole cells or
purified DNA (100 ng), 5 U Taq polymerase (Promega) in
1× PCR buffer (10 mM Tris–HCl [pH 8.4], 50 mM KCl, 2 mM
MgCl2 , 0.01 mM dNTPs, and 2 pmol/␮l primers) made up
to a final reaction volume of 25 ␮l with sterile water. The
PCR was carried out in a GeneAmp® PCR System 9700
(Applied Biosystems). Following 5 min of denaturation at
94 ◦ C, samples were subjected to 15 cycles in a TD pro-
gram (94 ◦ C for 30 s, 61 ◦ C for 30 s and 72 ◦ C for 45 s,
followed by a 0.5 ◦ C decrease of the annealing tempera-
ture every cycle). After completion of the TD program, 25
cycles were subsequently performed (94 ◦ C for 30 s, 48 ◦ C
for 30 s and 72 ◦ C s for 45 s) ending with a 20 min exten-
sion at 72 ◦ C. The reaction mix (1 ␮l) containing amplified
DNA was added to each well in a 96-well plate containing
a mixture of 12 ␮l LIZ® orange size standard and 1000 ␮l
formamide. The samples were processed and analyzed
on the ABI 3130 genetic analyzer (Applied Biosystems).
Predicted size Observed size
229 bp 230 bp
374 bp 372 bp
307 bp 307 bp
325 bp 325 bp
263 bp 263 bp
Results were analyzed with GeneMapper software (ver-
sion 3.7), which automatically determines the size of the
fluorophore-labelled amplicons based on a comparison to
the LIZ® size standard.
2.5. Bioinformatics analysis
To determine the expected sizes of amplicons from this
parasite with each of the satellite primer pairs, the Muguga
genome sequence (Gardner et al., 2005; GenBank Acces-
sion Number AAGK01000009) was obtained from the NCBI
website, and analyzed using the BLAST algorithm (Altschul
et al., 1997) and DNAMAN software.
3. Results
3.1. Capillary flow genotyping of T. parva DNA
A high-throughput satellite typing system was estab-
lished to characterize T. parva parasites. Five satellite
markers (Table 1) were chosen to analyze the T. parva DNA
using capillary flow electrophoresis and fluorophore detec-
tion. A histogram showing illustrative results as obtained
for one marker (MS7) with DNA from a cloned Muguga
parasite is shown in Fig. 1A. As the T. parva schizont stage
is believed to be haploid, only a single allele is present in
each parasite. Thus, each parasite type is expected to yield a
single PCR product for each marker. To determine the accu-
racy of this method, DNA from piroplasms derived from the
cloned sporozoite stabilate used in generating the T. parva
Muguga genome sequence was analyzed and the observed
amplicon sizes were compared to those predicted from the
genome sequence (Table 1). The observed sizes are simi-
lar to the predicted sizes. The alleles found in the cloned
T. parva Muguga DNA were labelled as ‘a’ and additional
peaks observed during the study were assigned subsequent
letters (Table 2).
3.2. Allelic diversity in the vaccine stabilates
DNA was extracted from each of the vaccine stabilates
and genotyped using the five satellite markers. As an exam-
ple, the histograms obtained with MS7 are shown in Fig. 1B
and C. The histograms obtain for FAO 1 and ILRI 0804 were
identical. Three peaks were detected in both stabilates, sug-
 E.H. Patel et al. / Veterinary Parasitology 179 (2011) 62–68 65

Fig. 1. Histograms showing alleles from piroplasm DNA extracted from an animal infected with the Muguga cloned sporozoite stabilate 3308 (A), ILRI 0804
vaccine stabilate (B) and FAO 1 vaccine stabilate (C), analyzed with MS7. The estimated size of each PCR amplicon is indicated below the peak.

gesting that the parasite populations in both stabilates have
three different MS7 alleles. The estimated product sizes are
288, 310 and 373 bp, the last of which is close to the pre-
dicted size of allele in T. parva Muguga cloned parasite (374
bp, Table 1). The genotyping results for all five markers are
summarized in Table 3, and indicate that there were no
detectable differences between the two vaccine stabilates.
Three different alleles of MS19 and MS25 were observed in
the stabilates, while ms9 and MS39 were represented by
two alleles each.
3.3. Allelic diversity in the reference stabilates
The reference stabilates were genotyped with the five
satellite markers, as shown in Table 3. The results indi-
cate that for both Muguga and Kiambu 5, there were
no detectable differences in allelic composition between
the FAO 1 and ILRI 08 reference stabilates, whereas
the Serengeti-transformed stabilate from ILRI 08 showed
greater allelic diversity than that from FAO 1. It also
appears that both Muguga reference stabilates and the
66 E.H. Patel et al. / Veterinary Parasitology 179 (2011) 62–68

Table 2 produced in vitro and analyzed with the five satellite mark-
Designation of mini- and microsatellite alleles based on observed ampli-
ers.
con size in capillary flow electrophoresis.

Allele ms9 MS7 MS19 MS25 MS39

3.4.1. Analysis of clones generated from ILRI 08 reference
a 231 373 307 325 263 stabilate
b 316 310 298 310 213
The numbers of clones analyzed and the genotyp-
c 288 323 253
ing results are summarized in Table 4. Fifty-two clones
obtained from the Muguga reference stabilate were ana-
Serengeti-transformed stabilate from ILRI 08 comprise lyzed, and eight different genotypes were identified. Four
more than one parasite population, whereas there is no evi- of these T. parva genotypes represent the major compo-
dence for more than one population in the other stabilates. nents within the stabilate, with a minimum of seven clones
The results also indicate that the Serengeti-transformed (≥13%) for each genotype. There are at least four minor
stabilates contain alleles that are also present in Muguga components, of which only two or three (≤6%) clones were
and Kiambu 5, whereas the Muguga and Kiambu 5 stabi- obtained. It was noted that the predominant clone type
lates do not appear to contain common alleles. had the same genotype as that of the cloned Muguga par-
asite from which the initial T. parva genome sequence
3.4. Analysis using in vitro cloning of the individual was obtained. The Serengeti-transformed reference stabi-
reference stabilates late was also shown to contain at least eight genotypes,
although there appeared to be only two major genotypes
In order to understand the complexity of the stabilates, which comprised 45% and 42% of the clones analyzed. These
cloned cell lines from each of the reference stabilates were genotypes are also present in the Muguga stabilate as one

Table 3
Mini- and microsatellite alleles identified in the vaccine and reference stabilates of FAO 1 and ILRI 08.

Stabilates ms 9 MS 7 MS 19 MS 25 MS 39

Vaccine
FAO 1 a+b a+b+c a+b+c a+b+c a+b
ILRI 0804 a+b a+b+c a+b+c a+b+c a+b
Reference
Muguga (F)a a a+b a+b a a
Muguga (I) a a+b a+b a a
Serengeti-transformed (F) a a b b a
Serengeti-transformed (I) a a +c b+c a+b a+b
Kiambu 5 (F) b c c c b
Kiambu 5 (I) b c c c b
a
F and I refer to the FAO 1 and ILRI 08 reference stabilates, respectively.

Table 4
Genotypes of T. parva identified in the Muguga, Serengeti-transformed and Kiambu 5 reference stabilates of FAO 1 and ILRI 08 by mini- and micro-satellite
analysis of cloned cell lines.

Genotypes ms9 MS7 MS19 MS25 MS39 ILRI 08 FAO 1

Muguga
1 a a a a a 15 (29%) 13 (33%)
2 a b b a a 13 (25%) 6 (15%)
3a a a b a a 7 (13%) 8 (21%)
4 a b a a a 7 (13%) 3 (8%)
5b a a b b a 3 (6%) 2 (5%)
6 a b b b a 3 (6%) 3 (8%)
7 a a a b a 2 (4%) 2 (5%)
8 a b a b a 2 (4%) 2 (5%)
52 39

Serengeti-transformed
1b a a b b a 30 (45%) 1 (25%)
2a a a b a a 28 (42%) 3 (75%)
3 b c b c b 3 (4%)
4 a a b c a 2 (3%)
5 a a c b a 1 (1.5%)
6 b c c b b 1 (1.5%)
7 b c c a b 1 (1.5%)
8c b c c c b 1 (1.5%)
67 4

Kiambu 5
1c b c c c b 52 (100%) 10 (100%)
a,b,c
Genotypes which are identical are indicated with the same superscript.
 E.H. Patel et al. / Veterinary Parasitology 179 (2011) 62–68
major (13%) and one minor (6%) component. All 52 clones
derived from the Kiambu 5 reference stabilate contain a
single genotype of T. parva detectable with these markers,
which is in line with the results obtained with the bulk ref-
erence stabilate. This genotype was also present as a minor
component of the Serengeti-transformed stabilate.
3.4.2. Analysis of clones generated from FAO 1 reference
stabilates and comparison with ILRI 08
Thirty-nine clones were genotyped from the Muguga
reference stabilate of FAO 1. The results indicate that there
are at least eight genotypes, all of which were found in the
ILRI 08 Muguga stabilate (Table 4). There were minor dif-
ferences in the relative proportion of the clones in each
stabilate. After six cloning attempts, only four clones were
obtained from the Serengeti-transformed reference stabi-
late. It may be possible that the sporozoites in this stabilate
have lost viability due to being stored in liquid nitrogen for
more than 12 years. Only two genotypes were identified
from this stabilate, which are the same as the major compo-
nents identified in ILRI 08 Serengeti-transformed reference
stabilate. The 10 clones derived from the FAO 1 Kiambu 5
reference stabilate displayed an identical genotype to those
derived from the ILRI 08 Kiambu 5 reference stabilate.
4. Discussion
The Muguga Cocktail is produced by inoculating three
seed stabilates separately into cattle. The seed stabilates
have been isolated in the laboratory from field ticks and
maintained by serial cattle-tick passage. The presence of
antigenic heterogeneity among T. parva parasites suggests
that the broad protection induced by the Muguga Cocktail
is dependent on there being heterogeneity in the protec-
tive antigens in the vaccine stabilate. Until these antigens
are identified, it is important that protective antigenic het-
erogeneity is maintained in the vaccine. This can only be
achieved through knowledge of the composition of suc-
cessive batches of the vaccine and seed stabilates and
comparison with previous batches and through protection
afforded in cattle.
The results of the present study show that the Muguga
Cocktail vaccine stabilate comprises at least 14 differ-
ent parasite types and is more complex than previously
reported. When analyzed with five satellite markers, the
diversity in parasite types resides in the Muguga and
Serengeti-transformed components of the stabilate with
only a single genotype being observed in the Kiambu 5
parasites. No major difference was observed in the com-
position of the vaccine stabilates made 12 years apart
which were both produced from the same seed stabi-
lates. In all, the results suggest the reference stabilates
derived from each seed stabilate appear to be composed
mostly of parasites unique to those stabilates, although
the Serengeti-transformed stabilates share one and two
genotypes with the Kiambu 5 and Muguga stabilates,
respectively. The genotypic analysis of the reference sta-
bilates suggests that no single genotype is present in all
three seed stabilates.
The only previous genotypic analysis of an extensive
series of clones derived from a T. parva Marikebuni sta-
67
bilate pointed towards the emergence of a dominant clone
after successive in vitro passages (Katzer et al., 2006). We
have, however, found no evidence of clonal dominance in
two of the three component stabilates of the Muguga Cock-
tail. In contrast, the third component stabilate, Kiambu 5,
is homogeneous with respect to the five loci that were
typed. Without the availability of the original isolates from
which the seed stabilates were derived, it is difficult to
determine if the current diversity represents gain or loss of
parasite types through cattle-tick passage. Gain of parasite
types may occur through genetic reassortment of alleles.
For example, it is possible that the original Muguga isolate
contained two types, one uniformly displaying ‘a’ alleles
and the other ‘b’ alleles and that the current mixture of
genotypes is the result of reassortment of ‘a’ and ‘b’ alleles.
Conversely, the original Kiambu 5 isolate may have been
isolated from an animal infected with a single parasite type,
or the current composition may reflect the emergence of a
dominant type.
The reference stabilates made 12 years apart from the
same seed stabilates displayed similar genotypic compo-
sition, although the lack of clones available for the earlier
Serengeti-transformed stabilate hampered a full analysis.
From the perspective of consistent vaccine production, it
was reassuring to observe that both Muguga reference sta-
bilates contained all eight genotypes. Nevertheless, these
results do not indicate if there is any functional differ-
ence between the vaccine stabilates. In immunization and
challenge experiments, we have observed that animals vac-
cinated with the ILRI 0804 stabilate were protected against
challenge with both the homologous and the FAO stabi-
late(s), which suggests that important protective antigens
have been conserved (unpublished observations). This can
only be fully assessed by sequencing the antigens known to
be important in inducing the protective immune response
following vaccination. Although we are currently sequenc-
ing 10 of the known CTL antigens to assess the amount of
diversity they display, it is not known whether these are
the only or even the major antigens that mediate protec-
tion.
These results will also provide more precise analysis
to address the field use of the ITM vaccine. Unimmunized
cattle co-grazing with ITM immunized cattle can be more
accurately monitored for the presence of parasites from the
vaccine, as previous studies have suggested (Oura et al.,
2007). In addition, the results will allow more detailed
studies to determine whether or not a prolonged carrier
state exists in the immunized animals and whether this
is due to vaccine or field parasites, or both. The infor-
mation on the composition of the vaccine will also be
important if there are any incidents of vaccine break-
through, where immunized cattle suffer from ECF due to
their being infected by a parasite sufficiently antigenically
distinct from those in the vaccine. However, since 2008
over 361,000 cattle have been immunized with the Muguga
Cocktail and there have been no reports of any break-
through stocks (Di Giulio et al., 2009).
The present study highlights the importance of geno-
typing clones derived from the vaccine stabilates, rather
than the bulk stabilates themselves or bulk cell lines
derived from them, to obtain a more complete assessment
68 E.H. Patel et al. / Veterinary Parasitology 179 (2011) 62–68
of the level of genotypic diversity. This may explain the dif-
ference between our results and those of Oura et al. (2007)
who observed only six genotypes in the vaccine stabilates.
A limitation of this approach is that the analysis is influ-
enced by the efficiency with which individual parasites can
infect and transform lymphocytes in vitro and the extent of
culture to obtain clones for the assay. It is not known if
more than one sporozoite can infect a single lymphocyte
and result in a mixed parasite population within the cell.
Katzer et al. (2006) used an in vitro cloning procedure and
addressed the possibility of satellite hypermutation follow-
ing infection and cloning. Our findings show that no mixed
genotypes were observed on the histograms from the indi-
vidual cell lines, suggesting that there were no satellite
hypermutations and that multiple sporozoite infections of
lymphocytes had not occurred, despite the high numbers
of sporozoites used.
5. Conclusions
The use of high-throughput capillary electrophore-
sis using a set of five satellite markers has revealed
that the Muguga Cocktail ITM vaccine stabilate is
more genotypically diverse than previously recognized.
Nevertheless, two batches of the vaccine stabilate
produced 12 years apart from the same seed stabi-
lates are genotypically similar. The results also show
that a full assessment of genotypic diversity requires
analysis of cloned cell lines, as minor components
are not discernible by analysing DNA derived from
whole stabilates. The use of these five satellite mark-
ers with capillary electrophoresis has proven to be
reproducible and a useful tool for identifying the com-
position of vaccine and reference batches and also in
field evaluations of these vaccines. While demonstrat-
ing complexity within the vaccine in certain regions
of the genome, this does not imply differences in the
capacity to induce protective responses in cattle. Stan-
dard procedures to produce and test vaccine stabilates
are providing vaccine stabilates of similar protective
quality.
Conflicts of interest
The authors have no known conflict of interest.
Acknowledgement
This is ILRI publication number IL-201004.
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