Best (Review) Babesiosis and Anaplasmosis

Veterinary Parasitology 180 (2011) 109–125
Contents lists available at ScienceDirect
Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar
Emerging perspectives in the research of bovine babesiosis and

anaplasmosis
Carlos E. Suarez ∗ , Susan Noh
Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, WA 99164-6630, USA
a r t i c l e i n f o a b s t r a c t
Keywords: The Babesia bovis and B. bigemina apicomplexan protozoa in conjunction with the rickettsia
Apicomplexan Anaplasma marginale are intraerythrocytic pathogens that are responsible for the most
Babesia bovis
prevalent and costly tick borne diseases (TBD’s) of cattle worldwide. These organisms are
Babesia bigemina
historically associated as they can cause clinically related hemolytic diseases in cattle, are
Anaplasma marginales
Vaccines all transmitted by Rhiphicephallus (Boophilus) ticks, and share an uncanny ability to evade
the immune systems of the vertebrate hosts, causing persistent disease. In addition, acute
babesiosis and anaplasmosis can be prevented quite effectively by combining tick control
and vaccination with living attenuated organisms. However these methods of control have
numerous limitations and improved approaches are needed. Importantly, immunizations
of cattle with inactivated experimental Babesia and Anaplasma vaccines can elicit variable
degrees of protection, indicating the feasibility for the development of inactivated or sub-
unit vaccines. A new research toolbox that includes full genome sequencing combined
with the improved ability to genetically modify the organisms is enhancing our under-
standing of their biology. An emerging paradigm is the use of recently developed Babesia
and Anaplasma transfection methods for functional gene characterizations and for vaccine
development. Promising recently identified subunit vaccine candidates are also emerg-
ing, including babesial proteases, putative rhoptry, microneme, and sexual stage antigens,
as well as subdominant, conserved, A. marginale outer membrane major surface proteins.
However, significant knowledge gaps on the role of key parasite molecules involved in
cell invasion, adhesion, asexual and sexual reproduction, tick transmission, and evasion
of the immune system, remain. A better understanding of the biology of these organisms
and the protective immune responses will positively contribute toward the goal of devel-
oping improved immunological and pharmacological interventions against these elusive
pathogens that are responsible for the most devastating TBD’s of cattle. Importantly, the
currently available research toolbox provides basic research instruments for helping close
current knowledge gaps which will aid the design and production of effective vaccines and
alternative pharmacological interventions.
Published by Elsevier B.V.
1. Introduction In particular, Babesia parasites and Anaplasma rickettsia,

are responsible for important diseases of large economic,
Tickborne diseases (TBD) are important factors limit- social, and epidemiological impact (De Vos, 1992; De
ing the development of livestock industries worldwide. Castro, 1997; Bock et al., 2004; Nari, 1995).
Although Babesia parasites are apicomplexan protozoa
and Anaplasma marginale is a rickettsia, they were histor-
∗ Corresponding author. Tel.: +1 509 335 6341; fax: +1 509 335 8328. ically linked since they form part of a complex of diseases
E-mail address: [email protected] (C.E. Suarez). also known as cattle tick fever, sharing important features:
0304-4017/$ – see front matter. Published by Elsevier B.V.

doi:10.1016/j.vetpar.2011.05.032
110 C.E. Suarez, S. Noh / Veterinary Parasitology 180 (2011) 109–125
Table 1
Babesia bovis and B. bigemina antigens currently investigated for the development of novel methods of control of bovine babesiosis.
Molecule/parasite Putative function – features Stage-localization References
RAP-1, B. bovis, RAP-1 a,b,c B. bigemina Erythrocyte binding, Blood and tick stages: Suarez et al. (1991, 1993, 1998,
invasion–neutralization sensitive; rhoptry–merozoites and 2003), Dalrymple et al. (1993),
signal peptide; immunodominant; sporozoites; surface; punctate Yokoyama et al. (2006) and
conserved; surface exposed pattern Mosqueda et al. (2002a,b)
epitopes
BboRhop68, B. bovis Post-invasion function, egression? Blood stages: Baravalle et al. (2010)
Trans-membrane domains. intra-erythrocytic merozoites,
Immunogenic trophozoites
RAP-1 related antigen (RRA) B. bovis Rhoptry? Invasion? Signal peptide, Rhoptry? Merozoites Suarez et al. (2011) and Brayton
neutralization sensitive. et al. (2007)
Subdominant- lowly expressed
VMSA: Erythrocyte recognition and Blood and tick stages: Hines et al. (1989, 1992, 1995a,b)
MSA-1, MSA-2, a, b, c of B. bovis attachment? merozoites; sporozoites Suarez et al. (2000), Jasmer et al.
Gp45 of B. bigemina Neutralization sensitive; signal Surface exposed; merozoites; (1992a), Florin-Christensen et al.
peptide; immunodominant; sporozoites (2002) and Fisher et al. (2001)
variable (MSA2c more conserved);
GPI anchor; glycosilated
Bb-AMA-1; BbTRAP Invasion? Micronemes? Secreted? Gaffar et al. (2003a,b) and Torina
B. bovis, Bb-AMA-1 Neutralization sensitive, et al. (2010)
B. bigemina conserved. Immunogenic?
Signal peptide-transmembrane
domains
Spherical body proteins Parasitosphorous vacuole? Spherical body; erythrocyte Hines et al. (1995a,b), Dowling
SBP-1, 2, 3, 4 Food acquisition? cytoplasm; Merozoites et al. (1996), Jasmer et al. (1992b)
Immunogenic and Terkawi et al. (2011)
Bbo-MIC-1 Cell recognition, attachment and Blood stages. Micronemes? Silva et al. (2010)
B. bovis invasion? Neutralization sensitive; Merozoites
sialic acid binding domains;
conserved. Immunogenic
Bbo-6cys A, B, C, D, E, F Sexual stage development? Bbo-6cysF: blood stages. Silva et al. (2011)
B. bovis Neutralization sensitive; Sexual stage parasites?
conserved; immunogenic
Large multigene families (vesa, Persistent infection, evasion, Blood stages: surface of Allred (2001), Allred and
smorfs). B. bovis antigenic variation; infected infected erythrocytes (vesa), Al-Khedery (2006), Al-Khedery and
erythrocyte sequestration. and unknown (smorfs) Allred (2006) and Brayton et al.
Immunogenic (2007)
Bov57. B. bovis, T (B) equi, B. bigemina Syntenic to protective T. parva Blood and tick stages: Freeman et al. (2010)
antigen p67, conserved merozoites, sporozoites
Bovipain-2, B. bovis; B. bigemina? Conserved, immunogenic; Blood stages: cytoplasm of B. Mesplet et al. (2010)
Nutrition? bovis and of infected
erythrocytes
they both infect exclusively erythrocytes in the verte- sequencing of Babesia and Anaplasma organisms together
brate hosts causing a clinically related acute disease, and with the development of novel research tools allowing
they are transmitted by Rhiphicephallus (Boophilus) ticks, genetic transformation are providing new insights on their
often co-occurring in endemic areas. Control of these TBD’s genetics (Brayton et al., 2005, 2007; Felsheim et al., 2010;
worldwide is currently based in large part on the use of live Herndon et al., 2010; Suarez and McElwain, 2010). Yet,
vaccines that are in some cases trivalent (based in B. bovis, B. despite of these advances, the most important limitations
bigemina attenuated strains and Anaplasma marginale sub- for developing improved control tools against these are the
species centrale organisms), and on the use of acaricides numerous and significant gaps in our understanding of the
targeting their tick vectors (De Castro, 1997; De Vos, 1992; biology of Babesia and Anaplasma, and in the protective
Standfast et al., 2003; Benavides et al., 2000; Vidotto et al., immune responses that they can elicit in cattle.
1998). In addition, both Babesia and Anaplasma can cause In this review we briefly summarize our current under-
persistent infections by avoiding the bovine immune sys- standing of bovine babesiosis caused by B. bovis and B.
tem using antigenic variation (Barbet, 2009). bigemina, and anaplasmosis, describe some current gaps in
Babesiosis and anaplasmosis remain prevalent world- knowledge, and highlight some new perspectives emerging
wide, in part, because the current approaches available from recent advances in research.
for control have many important limitations, and need
to be improved. Significant factors currently affecting the 2. Bovine babesiosis
control of babesiosis and anaplasmosis include increased
resistance to acaricides by ticks (Rosario-Cruz et al., 2009; Cattle babesiosis is caused mainly by the tick-borne
Jonsson et al., 2008), and the numerous drawbacks of the apicomplexan parasites B. bovis, B. bigemina, and B. diver-
current live vaccines (De Waal and Combrink, 2006; De gens (which is prevalent in Europe and has zoonotic
Vos and Bock, 2000; Kocan et al., 2010). Whole genome potential). The geographic distribution of Babesia para-
C.E. Suarez, S. Noh / Veterinary Parasitology 180 (2011) 109–125 111
Fig. 1. Blood stages during the development of Babesia bovis parasites in bovine erythrocyte in vitro cultures: (A) a mature intracellular B. bovis merozoite
pair is shown on the left side of the Panel. (B) B. bovis free merozoite, and a free merozoite attaching to a bovine erythrocyte surface; (C) intracellular B.
bovis trophozoites; (D) B. bovis kinetes isolated from the hemolymph of Riphicephalus microplus infected with B. bovis.
sites corresponds with that of their tick vectors (Graham 3. Babesia life cycle: knowledge gaps, and
and Hourrigan, 1977), mainly Rhiphicephalus (Boophilus) opportunities for intervention
microplus and R (B) annulatus in tropical and subtropi-
cal regions worldwide (Chauvin et al., 2009). In addition, Babesia bovis and B. bigemina, exhibit a typical apicom-
other Babesia parasites of limited geographic distribution, plexan life cycle characterized by merogony, gametogony,
B. ovata and B. major, are also known to cause disease in cat- and sporogony; have erythrocytes as the only single cell
tle. Because B. bovis and B. bigemina are the most significant target in the bovine host; and are transovarially transmit-
species infecting cattle and, they will be the main focus of ted (Riek, 1966; Melhorn and Schein, 1984; Hunfeld et al.,
this review. Babesiosis caused by B. divergens was reviewed 2008; Chauvin et al., 2009).
in deep elsewhere (Zintl et al., 2003). Cattle babesiosis is The Babesia life cycle was recently reviewed in depth
an acute disease that becomes persistent in surviving ani- (Chauvin et al., 2009) however a brief revision of the life
mals (Goff et al., 2001; Trueman and Blight, 1978). The cycle will help to illustrate recent advances and some of the
most common clinical symptoms associated with acute dis- important knowledge gaps that remain. The known anti-
ease include fever, hemolytic anemia, anorexia, lethargy, gens briefly discussed here and currently presumed to be
hemoglobinuria, tachycardia, and icterus. B. bovis is known involved in at different stages of the cycle are also described
for causing more severe disease often resulting in cerebral in Table 1.
babesiosis, characterized by convulsions, hyperaesthesia, Both B. bovis and B. bigemina sporozoites enter the
and paralysis, concomitant with sequestration of parasites hosts with the saliva of the infected feeding tick larva
in brain capillaries. Common causes leading to death in (B. bovis) or nymph (B. bigemina). Sporozoites invade ery-
acutely infected cattle are shock and respiratory distress throcytes where they divide asexually by binary fission
(Brown and Palmer, 1999). to become merozoites. Free and mature intraerythrocyte
B. bovis merozoites are shown in Fig. 1, Panels A and in invasion, and thus are being considered as subunit vac-
B, respectively. The mechanisms of erythrocyte invasion cine candidates. B. bigemina invasion studies suggested that
by sporozoites remain unknown, however, key B. bovis the invading Babesia parasites form a short lived para-
molecules that are required for erythrocyte invasion such sitophorous vacuole (PV) upon invasion that, in contrast
as MSA-1, MSA-2, and RAP-1 (Table 1), are also expressed to Plasmodium, appears to disappear shortly after internal-
in surface of the sporozoites (Mosqueda et al., 2002a,b). ization (Potgieter and Els, 1977; Yokoyama et al., 2006). It
Upon release, merozoites invade new erythrocytes where was suggested that the PV dissolves soon after internal-
they transform into trophozoites (Fig. 1C) that divide by ization in B. bovis (Yokoyama et al., 2006), although the
binary fission (merogony) to produce a pair of merozoites occurrence of a parasitophorous vacuole-based network
that perpetuate the cycle of erythrocyte invasion upon or a parasite surface coat in B. bovis-infected erythrocytes
exiting the erythrocyte. Our understanding of the process was also proposed recently (Okamura et al., 2007). At least
of erythrocyte invasion by B. bovis merozoites remains four B. bovis spherical body proteins, SBP-1, SBP-2, SBP-
incomplete and was reviewed by Yokoyama et al. (2006). 3, and SBP-4 have been so far identified (Jasmer et al.,
A study using an in vitro culture invasion assay suggested 1992b; Hines et al., 1995a,b; Dowling et al., 1996; Ruef et al.,
that invasion of erythrocytes by B. bovis is likely Ca2+ 2000; Yokoyama et al., 2006; Terkawi et al., 2011), but their
dependent, and requires the function of an actin–myosin- biological roles remain unknown (Yokoyama et al., 2006).
based motor, thus following the target cell invasion pattern In addition, there are only two types of putative rhoptry
of other related apicomplexan such as Plasmodium and Babesia proteins identified so far, the members of the RAP-
Toxoplasma parasites (Franssen et al., 2003; Carruthers 1 family, identified in B. bovis, B. bigemina, and in other
and Boothroyd, 2007). Thus, invasion of erythrocytes by Babesia parasites (Suarez et al., 1991, 1993; Mishra et al.,
Babesia merozoites seems to involve an initial stage of 1992; Dalrymple et al., 1993; Machado et al., 1993), and
erythrocyte-parasite recognition (Fig. 1B), probably using the B. bovis BboRhop68 (Baravalle et al., 2010). The RAP-
a sialic-acid dependent mechanism followed by attach- 1 molecules are expressed in merozoites, sporozoites and
ment and re-orientation of the merozoites (Gaffar et al., other asexual stages, are able to bind erythrocytes and con-
2003; Yokoyama et al., 2006). It was postulated that tain neutralization sensitive B-cell epitopes, and have been
GPI-anchored, neutralization sensitive parasite proteins, a leading candidate for vaccine development (Yokoyama
members of the variable merozoite surface antigen (VMSA) et al., 2002, 2006; Brown and Palmer, 1999; Norimine
that are expressed uniformly in the surface of B. bovis et al., 2003). However, their ligands and their exact roles
merozoites, are involved at the attachment stage of inva- in the invasion/egression mechanisms remain uncertain.
sion (Hines et al., 1992; Jasmer et al., 1992a; Suarez et al., The B. bovis RAP-1 proteins are encoded by two identi-
2000; Florin-Christensen et al., 2002; Carcy et al., 2006; cal head-to-tail and tandemly oriented genes (Dalrymple
Yokoyama et al., 2006). The GPI anchored gp45 antigen, et al., 1993; Suarez et al., 1998). In B. bigemina, the rap-
a surface-exposed 45 kDa variable protein that was able 1 gene family is more complex, consisting of a total of 5
to induce protection against challenge with homologous rap-1a, 5 rap-1b, and 1 rap-1c genes organized head-to-tail
strain was also identified in B. bigemina (McElwain et al., tandemly (Suarez et al., 2003). They are all transcribed but
1987, 1991; Fisher et al., 2001). In addition, the possible role only the rap-1a genes appear to be translated. Yet, a novel
of the surface exposed, conserved, and neutralization sen- RAP-1 related antigen (RRA), highly similar to B. bigemina
sitive Bbo-MIC1 protein containing a putative sialic-acid RAP-1b, containing all the motifs that define the babesial
binding domain in this process should also be explored RAP-1 proteins have also been recently identified in B. bovis
further (Silva et al., 2010). As a result of re-orientation, the (Suarez et al., 2011). RRA is expressed in minute amounts in
apical end of the merozoite comes in close contact with the merozoites, contains neutralization sensitive epitopes, it is
surface of the erythrocyte. Similar to other apicomplexans, highly conserved among distinct strains, and in contrast to
the apical end of the Babesia merozoites is equipped with RAP-1, is a subdominant antigen in infected cattle. Inter-
organelles, such as micronemes, rhoptries, and spherical estingly, the recently identified 68 kDa BboRhop68 was
bodies that contribute with their secretions to the pro- detected in intraerythrocytic trophozoites and merozoites
cess of internalization of the parasites (Potgieter and Els, but not in free merozoites suggesting that it is differentially
1977; Sam-Yellowe, 1996; Yokoyama et al., 2006). Based on expressed and may have a functional role immediately
studies done on Plasmodium and Toxoplasma, it is hypoth- after B. bovis erythrocyte invasion, or erythrocyte egres-
esized that during invasion, the microneme contents are sion by the mature merozoites (Baravalle et al., 2010).
secreted first followed by the contents of the rhoptries and Thus far, the mechanism used by Babesia merozoites to
the spherical bodies, an organelle resembling the dense egress from erythrocytes remains completely uncharacter-
granules in Plasmodium parasites (Mital and Ward, 2008; ized, and may also be a potential target for control.
Carruthers et al., 1999). Importantly, B. bovis homologs of Sexual reproduction of Babesia parasites starts when
the AMA-1 (BbAMA-1) and TRAP (BbTRAP) (Gaffar et al., some intracellular trophozoites develop into gametocytes
2004a,b), and B. bigemina AMA-1 (Torina et al., 2010), that are ingested by the ticks during feeding. The ingested
that are known to be secreted by micronemes in Babesia gametocytes undergo several morphological changes in the
related organisms such as Plasmodium and Toxoplama, have midgut of the ticks (ray bodies) through undefined mecha-
also been identified (Table 1). The exact functional role nisms to become competent for sexual reproduction, which
and the ligands for these molecules remain unknown, but requires the fusion of male and female gametes to gener-
because BbAMA-1 and BbTRAP contain neutralization sen- ate zygotes (Melhorn and Schein, 1984; Riek, 1966). It is
sitive epitopes, they are believed to play a significant role possible that, similar to Plasmodium, the sexual forms of
the parasites express sexual stage specific molecules, but 4. Babesia causes acute and persistent infections in
they remain unidentified. Sexual stage proteins expressed cattle
in Plasmodium includes the 6-cys protein family, which
includes the sexual stage antigen s48/45 domains (Pradel, The prepatent period following inoculation of B. bovis
2007). Comparative genomic approaches allowed identifi- by tick larva is generally between 6 and 12 days, with
cation of a novel B. bovis gene family encoding proteins with peak parasitemia and the manifestation of other clinical
similarities to the Plasmodium 6cys protein family, termed signs reached 3–5 days thereafter (Bock et al., 2004). Yet,
the Bbo-6cys gene family containing six genes termed Bbo- B. bovis parasitemia remains low in acutely infected cattle
6cys-A to F (Table 1). The Bbo-6cys A to E genes are tandemly likely due to the sequestration of the parasite in capillar-
arranged as a cluster on chromosome 2 in the B. bovis ies. The disease can be fulminant causing high mortality
genome, whereas the Bbo-6cys gene F is located in a dis- in adult and cattle, but less than one-year-old cattle are
tal region in the same chromosome (Silva et al., 2011). At a more resistant. Both B. bovis and B. bigemina have the ability
minimum the Bbo-cys6 E gene is expressed in blood stage to cause persistent infection in the face of strong immune
parasites, and because it contains neutralization sensitive responses in cattle surviving the acute disease. The mech-
B-cell epitopes, it might be of functional relevance and pos- anisms involved in persistence may differ between these
sibly required for survival of the parasites, but the pattern two parasites. Whereas B. bovis merozoites residing in ery-
of expression of the remaining genes of this family and throcytes can be sequestered in blood capillaries (mainly
their functional significance remains unknown and require cerebral and kidney), parasite sequestration has not been
further investigations. found to occur in B. bigemina. B. bovis infected erythro-
The zygotes, that are likely diploid, invade digestive cytes that remain sequestered do not circulate in the blood
cells in the midgut of the ticks. Although it was reported and avoid phagocytosis by spleen macrophages, favoring
that B. bigemina meiosis occurs in the midgut upon zygote establishment of persistent infections (Allred, 2001; Allred
infection (Mackenstedt et al., 1995), it remains unclear et al., 2000; Al-Khedery et al., 2006; Hutchings et al., 2007).
where and how meiotic division occurs for B. bovis. Zygotes B. bovis parasites are able to remodel the surface of the
then transform into kinetes (Fig. 1D) that can gain access erythrocyte resulting in a change of its mechanical and
to the hemolymph in the haemocoel of the tick vectors, adhesive properties to their advantage (reviewed by Gohil
where they invade the eggs in the tick’s ovaries. This pro- et al., 2010). Ridges formed on the surface of infected
cess allows transmission of Babesia parasites to the next erythrocytes (O’Connor et al., 1999; O’Connor and Allred,
generation of ticks, a mechanism known as transovarial 2000; Allred, 2000) were found to contain proteins encoded
transmission. Once in the larvae, the kinetes invade the by the B. bovis variable erythrocyte surface antigen (vesa)
cells in the salivary gland where they become sporozoites. gene family. These ridges play a key role in cytoadhesion
An important expansion in the number of sporozoites of the parasitized erythrocytes to epithelial capillary cells.
occurs in the salivary glands through sporogony (Homer In addition, the VESA proteins can undergo rapid anti-
et al., 2000). Sporozoites are transmitted to the vertebrate genic variation, that likely occurs through segmental gene
hosts after at least 2 or 3 days following attachment of B. conversion of the vesa multigene family (Al-Khedery and
bovis-infected tick larva, whereas transmission occurs dur- Allred, 2006; Brayton et al., 2007). Therefore, expression
ing tick nymph stages for B. bigemina. The events occurring of vesa proteins may be central to B. bovis persistence by
in the B. bovis and B. bigemina sporozoites residing in the playing a dual role in adhesion and immune evasion lead-
salivary gland of the R. (B.) microplus ticks leading to the ing to the facilitation of parasite survival in the face of the
formation of infective sporozoites, and their relationship immune pressure of the hosts. The mechanisms involved in
with the tick feeding mechanisms are also not yet well the persistence of B. bigemina parasites in cattle has been
understood. less explored, and it remains unknown whether expression
In summary, an overview of the life of the Babesia life of variable gene families also play a role in persistence,
cycle suggests there are many opportunities for develop- or if other adhesion phenotypes are required for parasite
ing intervention strategies for improved control, but it also survival in the hostile milieu of the host’s strong immune
reveals the existence of many important knowledge gaps. responses. Interestingly, B. bigemina erythrocytes uniquely
Specifically, more research is needed on the differential display host’s IgM molecules on their surface (Echaide et al.,
expression and functions of parasite molecules required 1998). It remains unknown whether surface displayed IgM
during the different stages of parasite development, the plays a role in adhesion, but together with its lack of tis-
molecular mechanisms and signals involved in invasion of sue sequestration, this suggests that B. bigemina may use
tick cells and erythrocytes, the mechanisms of egression, distinct persistence mechanisms than B. bovis.
the molecular events involved in the transition of the para- Despite clearance by macrophages and strong antibody
sites to sexual stages, the process of sexual reproduction in response acting against the parasite, a few infected erythro-
the tick, the occurrence of recombination during the sexual cytes remain in circulation in persistently infected bovines
stage, the timing and mechanisms involved in the mei- which assures the maintenance of a pathogen reservoir for
otic division, and the processes involved in the sporozoite ongoing transmission (Howell et al., 2007).
maturation in salivary glands of the ticks. Also, understand-
ing of the mechanisms of sexual reproduction of Babesia 5. Current methods of control of bovine babesiosis
parasites, could lead to the development of novel control
methods designed to block B. bovis transmission by inter- The negative impact of bovine babesiosis has in endemic
ference with the development of the sexual stage. areas, dictates the need for implementing control mea-
sures. A key factor is that cattle between 3 and 9 months of attenuated phenotype when inoculated into spleen-intact
age do not manifest clinical babesiosis. Furthermore, ani- 3–9 month-old steers. Although several explanations have
mals in this age group can develop long term and strong been proposed (Callow et al., 1979; Carson et al., 1990; Kahl
immunity against homologous and heterologous parasite et al., 1982, 1983), the mechanisms involved in attenuation
strains upon challenge. The resistance of young animals through serial passage remain unknown. An ideal feature
to the disease (Trueman and Blight, 1978; Goff et al., of the Babesia parasites that are used in vaccines is that they
2001; Zintl et al., 2005) is the basis of enzootic stability, are not transmissible by ticks. Vaccine strains that are not
defined as the state where the relationship between host, infective for ticks have been reported such as the B. bigem-
agent, vector and environment is such that clinical disease ina G vaccine strain (Bock et al., 2004) and a B. bovis vaccine
occurs rarely or not at all (Bock et al., 2004), and also for strain produced in Argentina (Mangold et al., 1993). Nev-
the success of the strategy of vaccination with attenuated ertheless, at least in Australia, where vaccination has been
Babesia parasites in areas at risk. In contrast, Bos taurus cat- extensively used for many years, vaccination did not seem
tle older than one year, are usually highly susceptible to to be able to introduce Babesia infections into previously
Babesia, including the vaccine strains (Trueman and Blight, uninfected areas (Bock et al., 2004). Production of live vac-
1978). The occurrence of serious outbreaks of babesiosis cines requires the amplification of the resulting attenuated
in endemic areas is usually related to the breakdown of parasites either in controlled bovine donors or in in vitro
enzootic stability (Smith et al., 2000). Transition toward cultures (Callow et al., 1997). Live Babesia vaccines are usu-
endemic instability usually results in catastrophic conse- ally safely administrated to young 3–9 months old steers,
quences for the herds and can be caused by environmental, but older animals can still be susceptible to the vaccine
mainly climatic, and/or human factors, including inconsis- strain and have a higher risk of succumbing to severe acute
tent tick control programs that result in the reduction of the disease upon vaccination.
inoculation rates and low degrees of antibabesial immunity
in the herds. Therefore, the use of sensitive diagnostic tests 6. Searching for subunit vaccine targets
is important to determine the degree of immunity in a herd.
This can be achieved by the use of novel standardized ELISA Live vaccines have many drawbacks such as the require-
tests based on the detection of anti-RAP-1 antibodies (Goff ment of a cold chain, a short shelf life, and the potential for
et al., 2006, 2008) or antibodies against other immunodom- the transmission of concurrent pathogens and for rever-
inant and conserved antigens (Bono et al., 2008; Terkawi sion to virulence (Shkap and Pipano, 2000; Fish et al., 2008).
et al., 2011). Because of the shortcomings, there is still the need for addi-
Taking into consideration all of these factors, the con- tional research on the development of alternative safer and
trol of bovine babesiosis can be accomplished either by tick better defined live or subunit vaccines. The observations
management, immunization, anti-Babesia drugs, such as that: (i) cattle persistently infected with Babesia are gener-
diminazen (Berenil) and imidocarb (Bock et al., 2004), or ally resistant to re-infection with related strains, termed
by a combination of these approaches. However, as selec- concomitant immunity (Brown et al., 2006); (ii) immu-
tion of imidocarb-resistant B. bovis parasites can be induced nity following immunization with live, attenuated B. bovis
experimentally (Rodriguez and Trees, 1996), it is conceiv- vaccine is known to last after parasites have been elim-
able that inappropriate use of the babesicides may lead inated by drug treatment (Brown et al., 2006; Dalgiesh,
to emergence of drug-resistant Babesia strains in the field 1993); and (iii) immunization with killed parasites or par-
(Zintl et al., 2003). Also, both imidocarb and berenil are asite extracts can confer some level of protective immunity
associated with residue problems in the food chain (Zintl following homologous or heterologous strain challenge
et al., 2003; Mdachi et al., 1995), and because B. bovis and B. (Bock et al., 1992; Timms et al., 1984; Montenegro-James
bigemina vaccine strains might be more sensitive to babesi- et al., 1985; Pattarroyo et al., 1995), suggested that devel-
cides, drug residues remaining in treated animals may also oping alternative subunit vaccines is an achievable goal.
interfere with vaccination efforts (Combrink et al., 2002). Furthermore, immunization with purified native surface
A widespread method used to diminish the impact exposed proteins such as RAP-1a and GP45 (McElwain
of acute clinical disease, is vaccination with attenuated et al., 1991), and with purified rhoptries also resulted in
Babesia parasites. variable degrees of protection against acute clinical dis-
Babesia parasites vary in their virulence, ranging from ease upon challenge with virulent B. bigemina (Machado
mildly to highly virulent and Babesia strains are poly- et al., 1999). Development of subunit vaccines requires a
clonal and often contain subpopulations of parasites that more refined understanding of the nature of the protec-
differ in virulence (Cowman et al., 1984; Nevils et al., tive immune responses against Babesia parasites. Briefly,
2000; Dalrymple et al., 1992). The nature of the factors protective adaptive responses require ␥-interferon pro-
that determine the virulent phenotype of Babesia parasites ducing CD4+T cells, and the production of opsonizing IgG2
remains unknown and is currently being investigated. The and complement fixing IgG1 antibody (Brown and Palmer,
B. bovis attenuated parasites used in vaccines are derived by 1999). In addition, the spleen and innate immunity in gen-
serial and rapid passage of a virulent strain among 20–30 eral play an important role both by helping to control the
splenectomized steers. In contrast, B. bigemina attenua- spread of infection in the hosts, and to prime the adaptive
tion is usually performed by slow passages of a virulent protective immune response. The importance of the spleen
strain in spleen-intact steers (Callow et al., 1979; Shkap and is evidenced by the increased susceptibility of splenec-
Pipano, 2000). However, in both cases, the parasite popu- tomized animals to Babesia infection. The roles of the innate
lations resulting from the serial passages generally have an system and our state of the art knowledge on the nature of
protective immune responses were extensively reviewed be an appropriate antigen for testing this emerging vaccine
recently (Brown et al., 2006; Goff et al., 2010). approach.
The search for protective antigens was initially focused
on identification of surface exposed conserved Babesia 7. Perspectives for developing new preventive and
proteins of presumed functional relevance, and later, on therapeutic interventions against Babesia parasites
those that were able to stimulate CD+4 lymphocytes that
secrete ␥-interferon, and in other strategies, as reviewed At least two major recent research developments that,
recently (Brown et al., 2006). Briefly, rational approaches in conjunction with the availability of Babesia culture sys-
toward the development of blood stage Babesia subunit tems and novel genomic, transcriptomic and proteomic
vaccines were focused initially on the identification of sur- techniques, have great potential to impact vaccine devel-
face exposed, conserved, functionally relevant, and highly opment are: (a) the complete genome sequence of Babesia
antigenic proteins, such as the members of the Rhoptry parasites; and, (b) the availability of novel functional
Associated Protein-1 (RAP-1) of Babesia. However, despite genomics tools, including a transfection system for B. bovis.
their ability to stimulate T-helper cell ␥-interferon and In addition, other important recent advances on the char-
IgG, testing of full size recombinant B. bovis RAP-1 or acterization of the mechanisms of antigenic variation, the
of RAP-1 fragments containing defined T-cell epitopes adhesion properties of infected erythrocytes, the mecha-
in bovine immunization experiments failed to confer nisms of cell invasion were reviewed in deep elsewhere
significant protection upon challenge of experimentally (Barbet, 2009; Allred and Al-Khedery, 2006; Gohil et al.,
immunized cattle with virulent B. bovis parasites (Norimine 2010; Hutchings et al., 2007; Yokoyama et al., 2006).
et al., 2003). Another vaccine candidate, the variable Mero-
zoite Major Antigen-1 (MSA-1), expressed uniformly on 7.1. Availability of genome data
the surface of the B. bovis merozoites was also tested
in cattle immunization trials on the basis of the ability Availability of the genome sequence of Babesia spp.
of anti-MSA-1 antibodies to strongly inhibit development has improved our understanding of the biology of the
of the parasite in in vitro neutralization assay (Hines parasite. Full genome sequences greatly accelerated the
et al., 1992; Suarez et al., 2000). However, recombinant identification of novel vaccine and pharmacological inter-
MSA-1 also failed to provide protection upon challenge vention targets. The ∼8.2 Mbp B. bovis genome contains
with B. bovis virulent strains (Hines et al., 1995a,b). It at least 3671 protein coding nuclear genes distributed
was reported that vaccination in Australia of cattle with among four chromosomes (Brayton et al., 2007). The largest
a combination of three recombinant B. bovis antigens gene family found in the genome encodes the polymor-
that included a 38-kDa cysteine-rich protein designated phic variant erythrocyte surface antigen protein (vesa). Full
12D3, a 60 kDa rhoptry protein designated T21B4 and structural characterization of the vesa genes coupled with
also called Bv60 and rhoptry-associated protein 1 (RAP- the identification of the molecular basis and the structural
1), and a high molecular weight antigen designated 11C5, determinants of the mechanisms involved in cytoadhesion
confers some protection upon challenge, but this immuno- can facilitate the development of a vaccine strategy aimed
gen was not further developed (Wright et al., 1992). In at blocking cell adhesion. Full genome analysis also has
a recent subunit vaccine trial, it was shown that immu- identified novel gene families encoding other proteins of
nization of steers with combined recombinant MSA-2c, potential functional significance such as the smorf gene
MSA-1 and 12D3 was not able to prevent clinical symp- family (Brayton et al., 2007; Lau, 2009).
toms upon challenge but the antigens were able to elicit Easily available comparative genomic tools, such as
an immunological response that was sufficient to pro- Blast searches, also facilitate identification of candidate
tect steers from a mild virulent strain of B. bovis (Alvarez genes that encode for proteins of functional relevance or
et al., 2010). In addition, other members of the VMSA are known to be targets of protective immune responses in
family containing neutralization sensitive epitopes, such other fully sequenced related apicomplexans, such as the
as MSA-2b should also be explored (Dominguez et al., more studied Plasmodium and Theileria parasites. This strat-
2010). In conclusion, relatively few blood stage antigens egy allowed recent identification of previously unknown
have been tested as vaccines, suggesting that new vac- and un-annotated genes and genes families that have the
cine candidate antigens need to be identified, and many potential for vaccine and drug development (Table 1), and
other new avenues of subunit vaccine development using for developing research leading to a better understand-
the already available vaccine candidates remain yet to ing of the biology of these parasites (Suarez et al., 2011;
be explored. Thus far, subunit vaccine trials based on Silva et al., 2010, 2011; Baravalle et al., 2010). For exam-
immunodominant antigens have failed to stimulate pro- ple, comparative genomics led to the identification of p67,
tective immunity (Brown et al., 2006). An interesting a Babesia bovis gene syntenic to Theileria parva p67 that
emerging approach is the identification of subdominant, is expressed in blood and tick stage parasites (Freeman
rather than immunodominant, antigens for subunit vac- et al., 2010) (Table 1). In addition, the recently character-
cine development. Subdominant antigens may be better ized papain-like cysteine proteases involved in important
vaccine candidates because antigens against which par- metabolic pathways, are candidates for both vaccine and
asites allowed the host to mount an immune response drug development (Mesplet et al., 2010). Additionally,
(immunodominant) are likely unimportant for the survival genome data mining has allowed for the development of
of the organism (Brown et al., 2006). The recently iden- tandem repeat-based multilocus typing systems used to
tified subdominant B. bovis RRA (Suarez et al., 2011) could differentiate B. bovis geographic isolates (Perez-Llaneza
et al., 2010; Simuunza et al., 2011a,b), and the discovery the characterization of signal peptides, to study traffick-
of differential transcription of rRNA genes in B. bovis para- ing pathways, or to characterize mechanisms of secretion
sites in distinct environmental conditions (Laughery et al., used by the parasite. These studies could be performed
2009). by determining cellular localization of fluorescent molec-
Interestingly these approaches, combined with struc- ular reporters, such as GFP, fused to the signal peptides
tural chemistry studies, also proved to be essential in under investigation. In contrast, stable transfection system
the identification of free GPI molecules and the genes requires more complex plasmid constructs and the oblig-
encoding for enzymes involved in the assembly of GPI atory use of a selectable marker. Recent experiments with
anchored proteins in the B. bovis genome (Rodríguez et al., stable transfection constructs were designed for integra-
2010). Inhibition of the GPI-pathway using mannosamine tion into the elongation factor-1␣ (ef-1˛) locus of the B.
in in vitro cultures resulted in abrogation in the growth of bovis parasite by homologous recombination (Suarez and
B. bovis, suggesting that this pathway is essential for sur- McElwain, 2009). The ef-1˛ locus has an identical organiza-
vival of the parasites (Rodríguez et al., 2010). Therefore, tion in both, B. bovis and Plasmodium parasites consisting
these findings suggest that further studies on this newly of two identical head to head, tandemly arranged genes
identified metabolic pathway might lead to the discovery separated by intergenic regions containing strong and con-
of novel drugs or vaccines that might be effective to control stitutive promoters (Vinkenoog et al., 1998; Suarez et al.,
Babesia parasites. 2006). Research performed in Plasmodium demonstrated
The B. bovis genome sequence also facilitated the that replacing one of two ef-1˛ genes present in malaria
characterization of the B. bovis expression profiles. This parasites by homologous recombination using transfection
approach can facilitate the identification of candidate vac- techniques would not greatly affect parasite viability (Janse
cine antigens, help to characterize the profile of expression et al., 2003). These observations provided rationale for the
at different stages, and overall, improve our understanding use of the ef-1˛ locus as a target of integration for the devel-
of the biology of the parasite (Lau et al., 2007). opment of a B. bovis stable transfection system (Suarez and
Taken together, these newly discovered genes and McElwain, 2010; Janse et al., 2003). The successfully trans-
sequences exemplify the potential impact of the use fected parasites were selected with antibiotic blasticidin,
of the information contained in the B. bovis genome. and they were able to express abundant and intracel-
Genome data, complemented with the characterization lular, cytoplasmic, green fluorescent (GFP-BSD) protein
of protective immune responses and the analysis of the (Fig. 2A). Stable transfection systems have a wide range
structure and pattern of expression of genes encoding for of possible application in vaccine research and develop-
functionally relevant proteins facilitates research on new ment including: (1) helping to define virulence factors by
approaches for developing subunit vaccines, new drugs, gene knock-out and complementation techniques; (2) a
and our improved understanding of the biology of Babesia tool aiding the functional characterization of genes; (3) the
parasites. In addition to the genome data, the role of epige- production of viable and attenuated Babesia strains that
netic regulation in Babesia, that so far remains unexplored, are deficient in known virulence factors; (4) introducing
may also help us to better understand expression of certain antigenic molecular markers that may help to discriminate
phenotypes in these parasites. vaccinated from naturally infected animals; (5) produc-
ing Babesia strains that express protective tick antigens, or
7.2. Use of functional genomics tools in Babesia research that over-express differentially expressed or subdominant
Babesia antigens. Current research efforts are focused on
More than half of the known B. bovis genes remain the application of transfection as a delivery system of for-
un-annotated and the functional role and biological rele- eign antigens, such as protective tick antigens with the goal
vance of most, either annotated or un-annotated genes, is of achieving dual vaccines that can protect cattle against
still unknown. The use of transcriptomic, proteomic and clinical babesiosis and also interfere with transmission of
transfection approaches, in combination with other tech- the parasite by targeting protective tick antigens and B.
niques, will be needed for the identification of genes that bovis antigens that are exclusively or highly expressed in
are differentially expressed in distinct stages of the par- tick stages of the parasite.
asite life cycle. In addition, transfection methods, which
allow the incorporation and expression of foreign DNA 8. Bovine anaplasmosis
into Babesia, are also useful for characterizing gene func-
tion, for studying mechanisms of gene regulation by using Anaplasma marginale is the most prevalent tick-borne
gene knock-out and supplementation strategies, and for pathogen of cattle worldwide with endemic regions in
vaccine development. A recent review described the devel- North, Central, and South America, as well as Africa Asia,
opment and potential applications of Babesia transient and and Australia (Brayton et al., 2009). Ixodid ticks are the
stable transfection systems in more depth (Suarez and biological vector of A. marginale, while mechanical trans-
McElwain, 2010). Briefly, the transient transfection sys- mission can occur through fly bites and reuse of needles
tem has potential to be used for experiments requiring (Sonenshine, 1991). Once an animal is exposed to this
short term expression of the gene of interest, which in gen- pathogen acute infection develops, which is character-
eral are selectable markers (i.e. blasticin-gfp fusion gene) ized by fever, high levels of bacteremia (≥107 bacterial/ml
and/or reporter genes (i.e. luciferase, GFP) (Suarez et al., of blood), anemia, weakness, reduced growth and milk
2004, 2006; Suarez and McElwain, 2008, 2009, 2010). For production, abortion, and in some cases, death (Kocan
instance, transient expression systems can be used for et al., 2010). Given time the acute infection is controlled
Fig. 2. Expression of green fluorescent protein (GFP) in transformed parasites as detected by epifluorescence microscopy. (A) Babesia bovis transformed
with the gfp-bsd gene (parasite line 1-2-124) expressed from the ef-1␣ locus of B. bovis (Suarez and McElwain, 2009). Localization of the expressed
chimeric protein was enhanced in formaldehyde-fixed cultured cells using an anti-GFP antibody and Alexa Fluor® 488 conjugated secondary antibody.
Expression of chimeric protein (green) is evident within the cytoplasm of two intracellular merozoites whose nuclei have also been stained by DAPI (blue).
Two autofluorescent non-infected cultured red blood cells are also visible (left panel = gfp immunofluorescence, middle panel = DAPI, right panel = merged
images). (B) Green fluorescent colonies of the St. Maries strain of A. marginale transformed with gfp in the midgut of a Dermacentor andersoni adult male
tick after 7 days of acquisition feeding.
and persistent infection develops, which is thought to terial numbers and ameliorating disease, the efficacy in
last for the life of the animal (Smith et al., 1989). Dur- clearing infection and thus preventing the establishment of
ing persistent infection, A. marginale is maintained below a pathogen reservoir is variable. Recently, treatment of per-
microscopically detectable levels which fluctuate between sistently infected steers that with oral chlortetracycline at
103 and 106 A. marginale/ml of blood, and overt clinical doses between 4.4 and 22 mg/kg per day for 80 days cleared
signs are generally absent (Eriks et al., 1993; French et al., A. marginale infection (Reinbold et al., 2010). The absence
1998; Kieser et al., 1990). Transovarial transmission of A. of infectious organisms was demonstrated by PCR by day
marginale from one tick generation to the next does not 49 of the treatment. Sub-inoculation of blood into splenec-
occur, thus the establishment of persistent infection within tomized animals was used to confirm complete clearance of
the bovine host is essential for ongoing transmission of this the organism. Discouragingly, animals cleared of infection
pathogen. through treatment were then re-infected with the origi-
nal strain (Reinbold et al., 2010). The results of treatment
9. Available control methods trials have been variable. In a second study, persistently
infected animals treated with 22 mg/kg of oxytetracycline
Currently, the tools available to prevent anaplasmo- intravenously every 24 h for 5 days failed to clear the infec-
sis are limited, and rely on the use of a live vaccine, tion (Coetzee et al., 2005). Additionally, long treatment of
antibiotic treatment, and the maintenance of endemic sta- animals with antibiotics on a large scale is becoming less
bility within a herd. The live vaccine, discussed in the acceptable as antibiotic resistance rises in both human and
next section, is not available in all parts of the world and animal pathogens. Given the significance of the disease
antibiotic treatment of clinically affected animals is diffi- burden, research started with the discovery of A. marginale
cult and expensive in extensive animal rearing systems. by Theiler (1910) in South Africa is being continued today in
Prevention of infection relies solely on tick control in the order to identify new means of disease control and preven-
face of increasing acaricide resistance among tick pop- tion. There are two likely targets of intervention: (1) at the
ulations (George et al., 2004; Rosario-Cruz et al., 2009). level of bovine immune system by induction of protective
While antibiotic treatment is effective in decreasing bac- immunity against A. marginale through vaccination and; (2)
less conventionally at the level of the tick, by preventing outer membrane proteins. Msp2 and Msp3, importantly,
tick colonization and transmission of A. marginale. are involved in immune evasion and establishment of
persistent infection though a mechanism of antigenic vari-
10. Identification of A. marginale vaccine targets ation. This process has been reviewed elsewhere (Palmer
et al., 2009). Briefly, Msp2 is expressed from a single expres-
In contrast to natural exposure, which often results in sion site, and is composed of a central hypervariable region
acutely high levels of bacteremia and anemia and long- that is flanked by highly conserved regions. Msp3 is thought
term persistence; immunization results in a significant to function similarly, though has not been examined as
reduction in bacteremia and anemia in nearly all animals extensively (Brayton et al., 2003; Futse et al., 2009; Meeus
and depending upon the immunogen, protection against et al., 2003). During infection within the host, the vari-
infection in 40–70% of immunized animals, providing a ation in the expressed version of Msp2 is generated by
rationale for vaccine development (Brown et al., 1998; gene conversion in which one of multiple msp2 donor alle-
Tebele et al., 1991; Noh et al., 2008). The mechanisms les is recombined into a single, operon-linked expression
of immune control of A. marginale are not completely site (Barbet et al., 2000; Brayton et al., 2001, 2002). Clear-
understood. However, protection against challenge asso- ance of a specific A. marginale Msp2 variant is accompanied
ciates with IgG2 directed toward outer membrane proteins by development of an antibody response directed toward
(Brown et al., 1998; Palmer et al., 1999; Tebele et al., 1991). that variant, indicating that Msp2, likely in combination
Two effective immunogens have been described; the first is with Msp3, is responsible for the ability of A. marginale to
A. marginale subsp. centrale, which is used as a live vaccine. evade the bovine immune response. Thus, Msp2 and Msp3
The second is composed of outer membrane proteins of A. have seemed likely vaccine targets. Experiments using both
marginale and has only been used experimentally (Bock and Msp2 and Msp3 as an immunogen have not been con-
de Vos, 2001; Pipano, 1995; Tebele et al., 1991). A. marginale ducted. However, immunization with native purified Msp2
subsp. centrale was isolated in S. Africa in 1911 (Theiler, containing a wide variety of variants did not confer protec-
1911). Soon thereafter protective immunity against viru- tion to cattle challenged with A. marginale expressing the
lent A. marginale challenge was demonstrated in animals same Msp2 variants as in the immunogen (Abbott et al.,
immunized with this organism (Theiler, 1912). A. marginale 2005). Additionally, antibody directed toward Msp2 corre-
subsp. centrale, while still used as a live vaccine in many lated with control of bacteremia infection, but not correlate
parts of the world has a high production cost, and carries with protection due to immunization (Noh et al., 2010b).
the risk of vaccine-induced disease as well as transmission Thus, the focus in identification of vaccine candidates has
of known and unknowns pathogens. Additionally, immu- shifted toward the identification of subdominant antigens
nity induced by live vaccination with A. marginale subsp. that confer protective immunity. This approach is ratio-
centrale is not uniform against all strains and outbreaks nal as subdominant antigens tend to be less variable. The
have been reported in immunized populations (Brizuela utility of this approach has been confirmed. For example,
et al., 1998; Turton et al., 1998). However, the overall effi- subdominant antigens have been identified that induce
cacy of this vaccine demonstrates that protection against neutralizing antibody to a variety of strains of the influenza
heterologous challenge in terms of disease prevention is virus (Ekiert et al., 2009; Kubota-Koketsu et al., 2009).
possible (Shkap et al., 2002). The protective outer mem- Through genomic and proteomic approaches, the
brane immunogen is derived from A. marginale isolated vaccine-relevant genome has been narrowed, and in par-
from erythrocytes. After lysis, the outer membrane fraction ticular, subdominant antigens have been identified using
of the isolated bacteria is concentrated using a sucrose gra- experiments linking vaccination/challenge trials with
dient. Consequently this immunogen is complex, laborious detailed proteomic analysis of the protective immuno-
to formulate, and thus is not appropriate for mass produc- gen. In one approach, the complex outer membrane
tion (Lopez et al., 2005; Tebele et al., 1991). Additionally, protein immunogen was separated into individual and
cross-protection among strains using the outer membrane small groups of components using 2-dimensional gel
immunogen has not been widely tested. Despite these con- electrophoresis. The immunoreactive proteins were then
straints, these two immunogens coupled with the complete identified using western blotting with sera from cattle
genome sequence of several strains of A. marginale serve as immunized with the protective A. marginale outer mem-
the foundation to identify the antigens relevant for induc- brane proteins. The immunoreactive proteins were excised
tion of broad, cross-protective immunity. and subjected to liquid chromatography followed by mass
The purified outer membrane of A. marginale which spectrometry for definitive identification by mapping to
induces protective immunity is composed of over 20 differ- the annotated genome (Lopez et al., 2005). This approach
ent proteins. The major surface proteins (Msps), including identified 24 immunodominant and subdominant anti-
Msp1, Msp2, Msp3, Msp4, and Msp5, which are all present gens which induced IgG2 production in the response to
in the outer membrane immunogen, were the first to be immunization. These included the previously character-
identified based on their abundance and their ability to ized major outer membrane proteins Msp2, Msp3, and
induce large amounts of antibody (Palmer and McGuire, Msp5, 21 newly described antigenic proteins, and multiple
1984). However, immunization with native, purified or housekeeping genes, which likely reflect the heteroge-
recombinant Msps generally leads to poor protection neous nature of the outer membrane protein immunogen.
(Palmer and McElwain, 1995). Of particular interest are To further narrow the potential vaccine targets and
Msp2 and Msp3 in part, because large amounts of anti- more closely link protective immunity with antigen identi-
body are directed toward these abundant, hypervariable fication, two approaches were taken. In the first approach,
intact A. marginale were isolated and treated with a mem- or vector molecules required for A. marginale to success-
brane impermeable cross-linking reagent, which resulted fully colonization and be transmitted from the tick.
in covalent linkage of a group of surface exposed outer Many species of ixodid ticks serve as competent vec-
membrane proteins. After purification, the protein com- tors of A. marginale (Kocan et al., 2010; Sonenshine, 1991).
plex was used to immunize cattle, and was analyzed by Most notable are members of the genus Rhipicephalus due
mass spectroscopy (Noh et al., 2008). This protein com- to their wide geographic distribution, overall debilitating
plex included only a subset of the complex outer membrane effects when present in high numbers on a host, and abil-
immunogen, but remained capable of the same significant ity to transmit Babesia spp. and A. marginale. Much of the
level of protection (Noh et al., 2008). Based on the pro- research concerning tick transmission of A. marginale has
teomic analysis, this protective complex contained 11 outer been done using Dermacentor spp., a primary vector of A.
membrane proteins: Omp1, Omp7–9, Msp1a, Msp2, Msp3, marginale in the United States, in part because of the size
Msp4, OpAG2, Am779, and the peptidoglycan-associated and hardiness of this tick in laboratory conditions. When
Am854 (Noh et al., 2008). comparing the ability of D. andersoni and R. microplus to
In the second set of experiments IgG2 from A. marginale transmit the Puerto Rico and Florida strains of A. marginale,
subsp. centrale immunized cattle was used to probe blots there were no differences in vector competence in terms
of A. marginale outer membranes separated in two dimen- of % infected ticks and pathogen transmission (Scoles et al.,
sions. The rational of these experiments was to identify 2007). Experiments using quantitative PCR to determine
antigens in A. marginale that are conserved in the protective the pathogen load in the tick organs have also been done.
vaccine strain (Agnes et al., 2011). In addition to Msp2 and In these experiments, the ability of D. andersoni and R.
Msp3, outer membrane proteins Omp7–9, 11, 13, and 14, microplus to transmit the St. Maries strain of A. marginale,
Am779, and AM854 were recognized. Excluding the hyper- isolated in the north western United States, and the Puerto
variable Msp2 and Msp3, conservation between the other Rico strain were compared (Futse et al., 2003). Both species
antigenic surface proteins of A. marginale and A. marginale of ticks were similarly able to acquire and transmit both
subsp. centrale ranged from 60 to 84%, indicating that a strains of A. marginale and both had similar infection rates
high degree of overall identity is not required to maintain (% infected ticks). The Puerto Rico strain replicated to
antigenic cross-reactivity. After excluding Msp2, and Msp3, higher levels in the salivary glands of R. microplus than
the two studies identified five outer membrane proteins in D. andersoni during transmission feeding, suggesting some
common, Omps 7–9, Am779 and Am854, indicating that degree of adaptation between the pathogen strain and its
pursuit of non-variable surface proteins may be rewarding available vector. While these two tick species perform sim-
in vaccine development. ilarly in laboratory conditions, the role they play in the
There is a gap in knowledge concerning the variability epidemiology of disease is unknown. In temperate regions,
of the outer membrane protein repertoire among popula- where Dermacentor spp. are the primary vector, the preva-
tions of A. marginale from geographically diverse regions. lence of A. marginale within infected herds tends to be
However, there is strong evidence suggesting that a subset relatively low. For example, 15% of animals in feedlots
of the vaccine candidates may be conserved and thus devel- in Iowa, located in the Midwestern United States, were
opment of a widely cross-protective vaccine is possible seropositive (Coetzee et al., 2010). In contrast, in tropical
(Junior et al., 2010). For example, Omp7 one of the vaccine regions, the prevalence tends to be quite high, from 37% in
candidates, has high % identity (86–99%) when comparing Costa Rica (Oliveira et al., 2011) to 58% in the Paraná state
the predominantly North American strains (Agnes et al., of Brazil (Marana et al., 2009) and Mbeere district of Kenya
2011). This identity drops to 65–72% when comparing (Gachohi et al., 2010), and up to 83% in some regions of
Omp7 from a Brazilian strain to the St. Maries and Florida Zambia (Simuunza et al., 2011a,b). There are likely many
strains, respectively (Junior et al., 2010). In contrast, many reasons for these differences, one of which may be the tick
other outer membrane proteins have a high % identity vector.
(92–100%) when comparing two N. American strains to a A. marginale strains with clearly different transmission
Brazilian strains. phenotypes have been characterized and serve as useful
tools to identify the genetic requirements of tick trans-
11. Mechanisms of tick transmission mission. For example, the St. Maries strain, which was
isolated from an animal in the Northwestern United States
A. marginale is an obligate intracellular pathogen, which suffering from severe anaplasmosis (PCV = 7%), is readily
displays marked host cell specificity. The transition from transmitted, with ≤10 Dermacentor andersoni ticks being
the bovine host to the tick is a dramatic shift in the sufficient for consistent transmission (Eriks et al., 1994;
host cell environment from the non-nucleated, metabol- Scoles et al., 2005). In contrast, A. marginale subsp. cen-
ically quiescent bovine erythrocyte to the metabolically trale has a low tick transmission efficiency phenotype. This
active, phagocytic and digestive tick midgut epithelial cell. organism infected fewer ticks, and replicated to signifi-
The pathogen must then traverse the hemolymph evading cantly lower levels in the tick midgut and salivary gland
the tick immune system in order to colonize the salivary as compared to the St. Maries strain (Ueti et al., 2009).
glands. Upon ingestion of a second blood meal, A. marginale Based on these quantitative differences as compared to
is transmitted to a new mammalian host. Understanding the high transmission phenotype of the St. Maries strain,
the molecular foundation of tick colonization and transmis- transmission of A. marginale subsp. centrale is estimated
sion presents largely unexplored opportunities for control to require >350 D. andersoni. This is supported by repli-
of this pathogen. Little is known about either the pathogen cate trials in which 70–250 adult male D. andersoni ticks
fed during either acute or persistent infection were unable spots, 15 A. marginale proteins, all annotated as hypothet-
to transmit A. marginale subsp. centrale (Agnes et al., 2010; ical were identified, including the previously identified
Galletti et al., 2009; Ueti et al., 2007, 2009) while successful Am778 as well as Am638, an ankyrin-repeat containing
transmission was observed with 425 adult male D. ander- protein. Additionally, many metabolic and housekeeping
soni (Ueti et al., 2009). Attempts to transmit A. marginale genes were identified, including, but not limited to dnaK,
subsp. centrale using Hyalomma excavatum, Rhipicephalus groEL, rpA. Two type 4 secretion system proteins were
sanguineus, and B. annulatus ticks all failed. However, less also identified, VirB10 and VirB11. Confirmation of the up-
than 200 ticks, well below the estimated number required regulation of a subset of these proteins, including Am470,
for transmission, were used in all cases (Shkap et al., 2009). Am410, and Am829 in tick cells as compared to bovine
At the far end of the spectrum are the apparently non- erythrocytes was done using western blotting and den-
tick transmissible strains. Despite various attempts with sitometry. Expression of these proteins in D. andersoni
several tick species, the Florida and Mississipppi strains midguts and salivary glands was verified using immuno-
have not been successfully tick transmitted (Smith et al., histochemistry. Of particular interest is the ankyrin-repeat
1986; Ueti et al., 2007). It has been hypothesized that these containing protein, Am638. Ankyrin repeats are highly con-
observed differences in tick transmission efficiency will be served motifs that are thought to mediate protein–protein
reflected in genetic differences among A. marginale strains. interaction and often localize to the host cell nucleus (Pan
More specifically, phenotypically distinct strains may dif- et al., 2008). Ankyrin repeat containing proteins from E.
fer in transmission efficiency due to strain-specific gene chafeensis and A. phagocytophilum, pathogens related to
content such that the presence or absence of a specific A. marginale, have been shown to traffic to the mam-
gene confers a tick transmission phenotype. Alternatively, malian host cell nucleus (Garcia-Garcia et al., 2009a,b; Zhu
a polymorphism in a gene may alter the function of the et al., 2009). Because A. marginale resides in non-nucleated
product thus leading to either increased or decreased trans- bovine erythrocytes during mammalian infection, proteins
mission efficiency. A third possibility is that differences which localize to the host cell nucleus would only be rele-
in regulatory regions may lead to increased or decreased vant during tick infection. It is unknown how these proteins
expression in genes which are common to both highly and actually function during A. marginale infection.
poorly transmitted strains (Agnes et al., 2010). While not all These proteomic approaches rely on differential expres-
of these possibilities have been addressed experimentally, sion of the respective gene products to infer functional
both comparative genomic and proteomic approaches have significance. While it is commonly hypothesized that pro-
been used to identify the molecular requirements of tick teins required for tick colonization are up-regulated during
transmission. tick colonization, additional constitutively expressed pro-
First, a comparative genomics approach was used to link teins may also be required. For example, many Mycobac-
specific gene content with the tick transmission phenotype. terium tuberculosis genes required for survival within
The genomes of the efficiently tick transmitted Virginia, the macrophage are not differentially regulated within
Puerto Rico, and St. Maries strains were compared to the the macrophage, but rather are constitutively expressed
non-tick transmitted Mississippi and Florida strains (Dark (Rengarajan et al., 2005). The genetic tools needed to
et al., 2009). Specific genes associated with transmission test functional requirement of both up-regulated and con-
phenotype were not identified (Dark et al., 2009). Thus, stitutively expressed proteins are in the early stages of
the differences in transmission phenotype are not due to development for Anaplasma spp. (Felsheim et al., 2006).
strain-specific gene content, but more likely due to dif- The St. Maries strain of A. marginale has been success-
ferences in shared genetic elements, either in coding or fully transformed with a green fluorescent protein (gfp)
regulatory regions. gene (Felsheim et al., 2010). The tick transmissibility, and
Two proteomics based approaches have been used to ability to establish persistent infection in the bovine was
identify proteins up-regulated during tick colonization recently demonstrated thus establishing the utility of this
(Ramabu et al., 2010; Noh et al., 2008). The first approach transformant as a model organism (Noh et al., 2010a).
involved comparing cross-linked surface exposed protein Further development of techniques to create targeted
complexes between A. marginale isolated from erythro- gene knockouts, and mutant libraries will be essential
cytes and ISE6 tick cells. Overall the variety of proteins for moving beyond association to identification of gene
expressed on the A. marginale colonizing the tick cells was function.
reduced to five as compared to fifteen identified on A.
marginale from bovine erythrocytes. Four proteins, Msp2,
Msp3, Msp4, and Am854, a peptidoglycan-associated pro- 12. Closing arguments
tein, were expressed in common. Am778 was the only
protein to be uniquely expressed on the A. marginale iso- While developing Babesia subunit vaccines might still be
lated from tick cells. These results were non-quantitative, an achievable goal, new tools will also allow for the produc-
and were not verified in A. marginale colonizing the tion of improved genetically modified live vaccines to be
tick vector. In a second quantitative approach, proteins developed using the framework of the current effective live
from A. marginale-infected and uninfected tick cells and vaccines. Thus, future improved live Babesia vaccines based
infected bovine erythrocytes were separated using two- on genetically attenuated organisms that can also function
dimensional gel electrophoresis and stained (Ramabu et al., as a delivery system for protective antigens expressed in
2010). Spots which were unique to infected tick cells were stages other than erythrocytic and/or from the tick vectors
submitted for mass spectrometric analysis. From those could be produced using transfection methods.
In addition, the two veins of anaplasmosis research transcription and transmission efficiency phenotype of Anaplasma
marginale. Infect. Immun. 78, 2446–2453.
summarized above are currently being mined with the goal
Al-Khedery, B., Allred, D.R., 2006. Antigenic variation in Babesia bovis
of developing effective tools to either induce immunity in occurs through segmental gene conversion of the ves multigene fam-
the bovine host through traditional methods of vaccina- ily, within a bidirectional locus of active transcription. Mol. Microbiol.
tion or to prevent transmission of A. marginale through, 59, 402–414.
Allred, D.R., 2001. Antigenic variation in babesiosis: is there more than
as yet, undiscovered methods of blocking tick transmis- one ‘why’? Microbes Infect. 3, 481–491.
sion. Rapid progress in vaccine development will require Allred, D.R., Al-Khedery, B., 2006. Antigenic variation as an exploitable
a better understanding of the correlates of immunity of weakness of babesial parasites. Vet. Parasitol. 138, 50–60.
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A. marginale, which can then be used to develop in vitro P.E., O’Connor, R.M., Stroup, S.E., 2000. The ves multigene family of B.
techniques to rapidly screen vaccine candidates. The devel- bovis encodes components of rapid antigenic variation at the infected
opment of tick transmission blocking tools will require the erythrocyte surface. Mol. Cell. 5, 153–162.
Alvarez, A.J., Lopez, U., Rojas, C., Borgonio, V.M., Sanchez, V., Castañeda,
ability to identify gene function, in particular to identify R., Vargas, P., Figueroa, J.V., 2010. Immunization of Bos taurus steers
genes required for tick colonization and transmission. Con- with Babesia bovis recombinant antigens MSA-1, MSA-2c and 12D3.
sequently, the ability to create targeted gene knock-outs Transbound Emerg. Dis. 57, 87–90.
Baravalle, M.E., Thompson, C., de Echaide, S.T., Palacios, C., Valentini, B.,
and whole genome mutant libraries of A. marginale will
Suárez, C.E., Christensen, M.F., Echaide, I., 2010. The novel protein
greatly facilitate this venture. BboRhop68 is expressed by intraerythrocytic stages of Babesia bovis.
Overall, the use of genome data in conjunction with Parasitol. Int. 59, 571–578.
Barbet, A.F., Lundgren, A., Yi, J., Rurangirwa, F.R., Palmer, G.H., 2000.
novel transfection and genomic related approaches will
Antigenic variation of Anaplasma marginale by expression of MSP2
be instrumental for closing our current knowledge gaps mosaics. Infect. Immun. 68, 6133–6138.
toward a better understanding of the biology of these Barbet, A.F., 2009. Persistence mechanisms in tick-borne diseases. Onder-
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Benavides, E., Vizcaino, O., Britto, C.M., Romero, A., Rubio, A., 2000.
therapeutic interventions. Together with a better under- Attenuated trivalent vaccine against babesiosis and anaplasmosis in
standing of the protective immune responses, these new Colombia. Ann. N. Y. Acad. Sci. 916, 613–616.
experimental approaches will likely be the keys leading to Bock, R.E., de Vos, A.J., 2001. Immunity following use of Australian tick
fever vaccine: a review of the evidence. Aust. Vet. J. 79, 832–839.
the developing of improved methods of control of these Bock, R.E., DeVos, A.J., Kingston, T.C., Sheils, I.A., Dalgliesh, R.J., 1992. Inves-
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Conflict of interest statement
Parasitology 129 (Suppl.), S247–S269.
Bono, M.F., Mangold, A.J., Baravalle, M.E., Valentini, B.S., Thompson, C.S.,
The authors state no conflict of interests. Wilkowsky, S.E., Echaide, I.E., Farber, M.D., Torioni de Echaide, S.M.,
2008. Efficiency of a recombinant MSA-2c-based ELISA to establish
the persistence of antibodies in cattle vaccinated with Babesia bovis.
Acknowledgments Vet. Parasitol. 157, 203–210.
Brayton, K.A., Dark, M.J., Palmer, G., 2009. Anaplasma. In: Nene, V.,
The authors would like to acknowledge the help of Kole, C. (Eds.), Genome Mapping and Genomics in Animal-associated
Microbes. Springer-Verlag, Berlin, Heidelberg, pp. 85–116.
Paul Lacy and Jacob Laughery, in the preparation of Brayton, K.A., Kappmeyer, L.S., Herndon, D.R., Dark, M.J., Tibbals, D.L.,
this manuscript and their help for preparing Fig. 1 and Palmer, G.H., McGuire, T.C., Knowles Jr., D.P., 2005. Complete genome
Table 1. Thanks to Dr. Christine Davitt, from the Franceschi sequencing of Anaplasma marginale reveals that the surface is skewed
to two superfamilies of outer membrane proteins. Proc. Natl. Acad.
Microscopy and Imaging Center of Washington State Uni- Sci. U.S.A. 102, 844–849.
versity for the pictures used in Fig. 1. Thanks to Dr. Massaro Brayton, K.A., Knowles, D.P., McGuire, T.C., Palmer, G.H., 2001. Efficient
Uetti, from ADRU-USDA-Agricultural Research Service, for use of a small genome to generate antigenic diversity in tick-borne
ehrlichial pathogens. Proc. Natl. Acad. Sci. U.S.A. 98, 4130–4135.
providing the picture of B. bovis kinetes. Thanks to Dr. David Brayton, K.A., Lau, A.O., Herndon, D.R., Hannick, L., Kappmeyer, L.S., Berens,
A. Schneider from ADRU-USDA-Agricultural Research Ser- S.J., Bidwell, S.L., Brown, W.C., Crabtree, J., Fadrosh, D., Feldblum, T.,
vice, and Marina Caballero who kindly provided the Babesia Forberger, H.A., Haas, B.J., Howell, J.M., Khouri, H., Koo, H., Mann, D.J.,
Norimine, J., Paulsen, I.T., Radune, D., Ren, Q., Smith Jr., R.K., Suarez,
bovis pictures shown in Fig. 2, and to Dr. Don Knowles C.E., White, O., Wortman, J.R., Knowles Jr., D.P., McElwain, T.F., Nene,
for his continuous and friendly support and for criti- V.M., 2007. Genome sequence of Babesia bovis and comparative anal-
cally reading the manuscript. This work was supported by ysis of apicomplexan hemoprotozoa. PLoS Pathog. 3, 1401–1413.
Brayton, K.A., Meeus, P.F., Barbet, A.F., Palmer, G.H., 2003. Simultaneous
USDA-ARS CRIS Project No. 5348-32000-028-00D, USAID
variation of the immunodominant outer membrane proteins, MSP2
grant PCE-G-0098-00043-00, and USDA (SCA58-5348-7- and MSP3, during Anaplasma marginale persistence in vivo. Infect.
528) agreement 5348-32000-028-055. Immun. 71, 6627–6632.
Brayton, K.A., Palmer, G.H., Lundgren, A., Yi, J., Barbet, A.F., 2002. Antigenic
variation of Anaplasma marginale msp2 occurs by combinatorial gene
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Veterinary Parasitology 180 (2011) 109–125

Contents lists available at ScienceDirect

Emerging perspectives in the research of bovine babesiosis and

1. Introduction In particular, Babesia parasites and Anaplasma rickettsia,

0304-4017/$ – see front matter. Published by Elsevier B.V.

Molecule/parasite Putative function – features Stage-localization References

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