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Pathogenicity of Theileria parva is influenced by the host cell type infected by the parasite

01 Feb 1996-Infection and Immunity (American Society for Microbiology)-Vol. 64, Iss: 2, pp 557-562

TL;DR: The results of these experiments indicate that the cell type infected by T. parva influences the pathogenicity of the parasite.

AbstractTheileria parva has been shown to infect and transform B cells and T cells at similar frequencies in vitro. However, the majority of parasitized cells in the tissues of infected cattle are alpha/beta T cells. The aim of this study was to determine whether the cell type infected with T. parva influenced the pathogenicity of the parasite. The initial approach, which involved inoculation of cattle with autologous cloned cell lines of different phenotypes, failed to resolve the issue, because of prolonged period of culture required to clone and characterize the cell lines resulted in attenuation of the cells. As an alternative approach, cattle were inoculated with purified populations of autologous cells that had been incubated in vitro with T. parva sporozoites for 48 h. As few as 3 x 10(4) peripheral blood mononuclear cells (PBMC) treated in this way were found to produce severe clinical reactions with high levels of parasitosis. Infections of similar severity were produced with purified populations of CD2+, CD4+, and CD8+ T cells. By contrast, infected B cells gave rise to mild self-limiting infections even when administered at a 10-fold-higher dose. In animals that received infected CD4+ or CD8+ T cells, the parasitized cells in the lymph nodes on day 11 of infection were all within the CD4+ and CD8+ populations, respectively, indicating that there had been minimal transfer of the parasite between cell types. Phenotypic analyses of cultures of PBMC infected in vitro with saturating concentrations of sporozoites revealed that parasitized B cells were abundant in the cultures after 1 week but were subsequently overgrown by T cells. The results of these experiments indicate that the cell type infected by T. parva influences the pathogenicity of the parasite.

Topics: Theileria parva (60%), CD8 (55%), Clone (B-cell biology) (53%), Cell culture (53%), Peripheral blood mononuclear cell (51%)

Summary (2 min read)

MATERIALS AND METHODS

  • The animals were produced on the International Livestock Research Institute ranch, which is free of T. parva, and were reared indoors under parasite-free conditions from 4 to 5 days of age.
  • The level of infection in each batch of ticks was estimated by counting the number of infected acini in a sample of dissected salivary glands stained with methyl green pyronine.
  • Purified populations of B cells and T cells were obtained by indirect immunofluorescence staining of PBMC with specific monoclonal antibodies (MAbs) and then by sorting of positive cells on a FACStar Plus cell sorter (Becton Dickinson, Mountain View, Calif.).
  • In each experiment, a sample of sorted cells was checked for purity and if necessary the cells were subjected to a second round of sorting.

Infection of cells with T. parva.

  • Infected tick salivary glands were suspended in the same medium and ground in a glass tissue grinder at room temperature.
  • To obtain cloned infected cell lines, cells were distributed at limiting dilutions ranging from 10 3 cells to 1 cell per well into 96-well plates containing a fibroblast feeder layer (BT6) derived from bovine fetal thymus tissue, as described previously (1) .
  • In some experiments, sorted cells infected with sporozoites were inoculated back into the donor animals after 48 h of culture in medium containing 2.5% T-cell growth factor derived from concanavalin A-stimulated PBMC as described previously (26) .
  • The inocula were administered subcutaneously on the right side of the neck, and animals were monitored daily for rectal temperature and the appearance of parasitized cells, from day 5 until the experiments were terminated.
  • The positive and negative cells in each stained population were sorted to Ͼ98% purity on a FACStar Plus cell sorter, and cytospin smears were prepared and stained by immunofluorescence for detection of parasitized cells in the same way as for lymph node puncture biopsy samples.

Reactions of animals inoculated with cloned autologous cell lines.

  • Previous studies had shown that inoculation of cattle with 10 5 or more autologous parasitized cells from recently established cell lines resulted in severe, often fatal infections (5) .
  • All of the animals were challenged in two groups, along with susceptible controls, with a lethal dose of T. parva sporozoites at 4 and 12 weeks after the initial infection.
  • Two of the animals that initially showed no reaction following inoculation with infected B-cell lines (C193 and C672) developed severe infections with prolonged fever and high levels of parasitosis in regional and contralateral lymph nodes.
  • Two of the animals exhibited fever for 1 day, and a few parasites were detected in the regional lymph node of only one animal (B312) for 3 days (data not Phenotypes of parasitized cells in animals infected with defined cell populations.
  • In the animal infected with whole PBMC, parasitized cells were found within both the CD4 ϩ and CD4 Ϫ populations, although a majority were CD4 ϩ .

DISCUSSION

  • Primary infections of cattle with T. parva are characterized by rapid multiplication of parasitized lymphoblasts which become disseminated throughout the lymphoid system and are responsible for much of the pathology associated with infection (16, 23) .
  • This suggested either that relatively few B cells became infected in vivo or that B cells and T cells infected with T. parva differ in their capacities to undergo unregulated growth in vivo.
  • The difference between the outcome of these infections and the outcome of those in their previous study (5) probably relates to the use of cloned cell lines which had been heavily selected for growth in vitro and had been maintained in culture for a longer period (10 to 16 weeks) to facilitate cloning and phenotyping.
  • Two of the four cattle that received parasitized B cell lines showed no clinical or parasitological reaction and were susceptible to subsequent challenge with sporozoites.
  • These observations indicate that the difference in outcome of infection in animals inoculated with infected B cells or T cells is related to differences in growth or regulation of the infected cells rather than merely reflecting the numbers of cells in which the parasite had established infection.

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Pathogenicity of Theileria parva is influenced by the host cell
type infected by the parasite
Citation for published version:
Morrison, I, MacHugh, N & Lalor, PA 1996, 'Pathogenicity of Theileria parva is influenced by the host cell
type infected by the parasite', Infection and Immunity, vol. 64, no. 2, pp. 557-62.
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INFECTION AND IMMUNITY, Feb. 1996, p. 557–562 Vol. 64, No. 2
0019-9567/96/$04.0010
Copyright q 1996, American Society for Microbiology
Pathogenicity of Theileria parva Is Influenced by the Host
Cell Type Infected by the Parasite
W. IVAN MORRISON,
1
* NIALL D. MACHUGH,
2
AND PAUL A. LALOR
3
Institute for Animal Health, Compton, United Kingdom
1
; International Livestock Research Institute,
Nairobi, Kenya
2
; and 35 Liston Street, Burwood, Victoria 3125, Australia
3
Received 1 August 1995/Returned for modification 21 August 1995/Accepted 16 November 1995
Theileria parva has been shown to infect and transform B cells and T cells at similar frequencies in vitro.
However, the majority of parasitized cells in the tissues of infected cattle are a/b T cells. The aim of this study
was to determine whether the cell type infected with T. parva influenced the pathogenicity of the parasite. The
initial approach, which involved inoculation of cattle with autologous cloned cell lines of different phenotypes,
failed to resolve the issue, because the prolonged period of culture required to clone and characterize the cell
lines resulted in attenuation of the cells. As an alternative approach, cattle were inoculated with purified
populations of autologous cells that had been incubated in vitro with T. parva sporozoites for 48 h. As few as
3 3 10
4
peripheral blood mononuclear cells (PBMC) treated in this way were found to produce severe clinical
reactions with high levels of parasitosis. Infections of similar severity were produced with purified populations
of CD2
1
, CD4
1
, and CD8
1
T cells. By contrast, infected B cells gave rise to mild self-limiting infections even
when administered at a 10-fold-higher dose. In animals that received infected CD4
1
or CD8
1
T cells, the
parasitized cells in the lymph nodes on day 11 of infection were all within the CD4
1
and CD8
1
populations,
respectively, indicating that there had been minimal transfer of the parasite between cell types. Phenotypic
analyses of cultures of PBMC infected in vitro with saturating concentrations of sporozoites revealed that
parasitized B cells were abundant in the cultures after 1 week but were subsequently overgrown by T cells. The
results of these experiments indicate that the cell type infected by T. parva influences the pathogenicity of the
parasite.
Theileria parva is a tick-borne protozoan parasite of cattle
that infects lymphocytes and causes an acute, usually fatal
lymphoproliferative disease known as East Coast fever. The
disease is characterized by the presence of large numbers of
parasitized lymphoblasts throughout the lymphoid system,
which eventually leads to lymphocyte destruction and leuko-
penia (reviewed in reference 15). A unique relationship of T.
parva with host lymphocytes is central to its ability to undergo
rapid replication in the animal. Following invasion of the lym-
phocyte, the sporozoite rapidly gains access to the cytosol and
develops into the multinucleate schizont, an event that is as-
sociated with activation and proliferation of the infected cell.
An association with the mitotic spindle enables the parasite to
divide synchronously with the lymphocyte (14). Thus, multipli-
cation of parasites is believed to occur mainly by clonal expan-
sion of the lymphocytes initially infected with sporozoites. This
characteristic of parasitized cells allows them to be propagated
in vitro as continuously growing cell lines; such lines can be
established with cells taken from infected cattle (20) or by
infection of lymphocytes in vitro with sporozoites (4).
Limiting-dilution cultures of purified populations of lympho-
cytes infected in vitro with sporozoites have been employed to
identify the cell types that support growth of the parasite.
These experiments have demonstrated that T cells of CD4,
CD8, or g/d lineages and B cells can all be infected at similar
frequencies (1). Phenotypic analyses of the resultant cloned
cell lines showed that each cell type retained a distinctive
phenotype, although expression of immunoglobulin was down-
regulated in infected B cells and many infected CD4
1
clones
coexpressed CD8. By contrast, analyses of cells from lymphoid
tissues of infected cattle showed that virtually all of the in-
fected cells expressed T-cell markers (11). An important ques-
tion raised by these findings is whether parasitized T and B
cells differ in their growth characteristics in vivo and hence in
their capacity to cause disease or whether there is preferential
infection of T cells in vivo. The studies described herein set out
to resolve these issues by examining the nature of infections
established following inoculation of cattle with purified popu-
lations of autologous lymphocytes infected with T. parva.
MATERIALS AND METHODS
Animals. All the animals used in the study were female or castrated male
Boran cattle (Bos indicus) between 6 and 18 months old. The animals were
produced on the International Livestock Research Institute ranch, which is free
of T. parva, and were reared indoors under parasite-free conditions from 4 to 5
days of age. All animals were seronegative for antibodies to T. parva schizonts
(13) at the outset of the experiments.
Parasites. The Muguga stock of T. parva was used throughout. Sporozoites
were harvested from salivary glands dissected from infected nymphal Rhipiceph-
alus appendiculatus ticks that had been prefed on rabbits for 5 days as described
previously (5). The level of infection in each batch of ticks was estimated by
counting the number of infected acini in a sample of dissected salivary glands
stained with methyl green pyronine.
Cell populations. Peripheral blood mononuclear cells (PBMC) were isolated
from venous blood by density gradient centrifugation as described elsewhere
(18). Purified populations of B cells and T cells were obtained by indirect
immunofluorescence staining of PBMC with specific monoclonal antibodies
(MAbs) and then by sorting of positive cells on a FACStar Plus cell sorter
(Becton Dickinson, Mountain View, Calif.). In each experiment, a sample of
sorted cells was checked for purity and if necessary the cells were subjected to a
second round of sorting. All sorted cell populations used in the experiments were
of 98% or greater purity. The following MAbs were employed: CH128A (im-
munoglobulin G1 [IgG1]), IL-A11 (IgG2a), and IL-A51 (IgG2a), specific for
bovine CD2, CD4, and CD8, respectively (2, 8, 19); IL-A30 (IgG2a), specific for
bovine IgM (27); and IL-A29 (IgG1), specific for a molecule termed WC1 whose
expression is restricted to bovine g/d T cells (6).
* Corresponding author. Mailing address: Institute for Animal
Health, Compton Laboratory, Compton, Nr Newbury, Berkshire
RG20 7NN, UK. Phone: 1635 578411. Fax: 1635 577263. Electronic
mail address: Samantha.Cook@BBSRC.ac.uk.
557

Infection of cells with T. parva. For infection with T. parva, PBMC and sorted
lymphocytes were suspended at 2 3 10
7
cells per ml in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal bovine serum,
L-glutamine, 5 3
10
25
M 2-mercaptoethanol, and gentamicin (culture medium). Infected tick
salivary glands were suspended in the same medium and ground in a glass tissue
grinder at room temperature. After centrifugation at 100 3 g for 5 min to remove
coarse debris, the sporozoite suspension was adjusted to a concentration esti-
mated to be the equivalent of 2,000 infected salivary gland acini per ml. Previous
studies had shown that this concentration of sporozoites gave saturating levels of
binding (i.e., maximal numbers of cells with sporozoites bound) to PBMC (17).
Equal volumes of sporozoites and cells were mixed in 10-ml polycarbonate test
tubes, and, after incubation at 378C for 90 min with periodic mixing, the cells
were centrifuged, washed once, and resuspended in culture medium at 4 3
10
6
/ml. For establishment of bulk cell lines, the cells were distributed either in
1-ml aliquots into 24-well cluster plates or in 5-ml aliquots into T25 flasks. To
obtain cloned infected cell lines, cells were distributed at limiting dilutions
ranging from 10
3
cells to 1 cell per well into 96-well plates containing a fibroblast
feeder layer (BT6) derived from bovine fetal thymus tissue, as described previ-
ously (1). Cell line establishment was monitored microscopically, and wells with
cell growth at a clonal frequency were expanded first into 24-well cluster plates
and then into T25 flasks. Cell lines were maintained by subculture and addition
of fresh culture medium every 2 to 3 days.
In some experiments, sorted cells infected with sporozoites were inoculated
back into the donor animals after 48 h of culture in medium containing 2.5%
T-cell growth factor derived from concanavalin A-stimulated PBMC as described
previously (26).
The phenotypes of infected cell lines were determined by flow-cytometric
analysis, on a FACStar Plus cell sorter, after staining by indirect immunofluo-
rescence with a panel of MAbs specific for bovine leukocyte differentiation
antigens as described elsewhere (18).
Infection of cattle. Two types of experiment were carried out in cattle. In the
first, a group of 11 cattle was inoculated each with 10
5
(in one animal 5 3 10
4
)
autologous parasitized cells from cloned cell lines of different phenotypes. (For
the origins and phenotypes of the cell lines, see Table 1.) They consisted of six
CD4
1
or CD8
1
cell lines derived by cloning from cell lines established by
infection of PBMC in vitro, one cell line obtained by infection and limiting
dilution cloning of purified g/d T cells (WC1
1
), and four cell lines obtained by
infection and limiting dilution cloning of purified B cells. Three of the four CD4
1
cell lines coexpressed CD8 on a variable proportion of cells. The cloned cell lines
had been in culture for periods ranging from 10 to 16 weeks at the time that they
were used to inoculate cattle.
The second experimental approach involved inoculation of cattle with autol-
ogous PBMC or purified populations of T or B lymphocytes, which had been
incubated in vitro with sporozoites for 48 h as described above. Because of the
time that was required to obtain purified populations of lymphocytes by cell
sorting, it was not possible to use large numbers of animals in a single experi-
ment. Therefore, a series of four experiments, each involving three or four
animals, was carried out (see Table 2).
Cells destined for inoculation into cattle were centrifuged, washed once in
culture medium, and resuspended in 1 ml of phosphate-buffered saline, pH 7.2.
The inocula were administered subcutaneously on the right side of the neck, and
animals were monitored daily for rectal temperature and the appearance of
parasitized cells, from day 5 until the experiments were terminated. Smears
prepared from puncture biopsy samples of the right and left prescapular lymph
nodes were fixed in cold acetone for 2 min and, after drying, stained with
fluorescein isothiocyanate-labelled bovine antiserum to T. parva and examined
microscopically for the presence of parasites. The percent parasitosis was deter-
mined by counting the number of schizonts in 1,000 cells.
All of the animals in the first experiment (see Table 1) and those that did not
succumb to infection in the second set of experiments (see Table 2) were
challenged, along with susceptible control animals, with a lethal dose of cryo-
preserved T. parva (Muguga) sporozoites (stabilate 836) between 4 and 12 weeks
after initial infection. The parasites were injected subcutaneously on the right
side of the neck and the animals were monitored clinically and parasitologically
on a daily basis as described above for primary infections.
Phenotypes of parasitized cells in infected animals. In two of the experiments
in which cattle were inoculated with purified populations of lymphocytes that had
been incubated with sporozoites (experiments 3 and 4 [see Table 2]), the phe-
notypes of infected cells in the lymph nodes were examined on day 11 after
infection. Lymph node puncture biopsy samples were collected into a 5-ml
syringe containing Alsever’s solution, and viable mononuclear cells were isolated
by centrifugation over Ficoll Isopaque/Pharmacia at 900 3 g for 20 min. The cells
were washed once in Alsever’s solution and stained by indirect immunofluores-
cence with MAb specific for T-cell populations. The positive and negative cells in
each stained population were sorted to .98% purity on a FACStar Plus cell
sorter, and cytospin smears were prepared and stained by immunofluorescence
for detection of parasitized cells in the same way as for lymph node puncture
biopsy samples.
RESULTS
Reactions of animals inoculated with cloned autologous cell
lines. Previous studies had shown that inoculation of cattle
with 10
5
or more autologous parasitized cells from recently
established cell lines resulted in severe, often fatal infections
(5). In an attempt to investigate the influence of the phenotype
of the infected cells on the severity of infection, 11 cattle were
each inoculated with 10
5
(in one animal 5 3 10
4
) autologous
parasitized cells from established cloned cell lines. The phe-
notypes of the cell lines and the clinical reactions of the ani-
mals following inoculation are presented in Table 1.
The severity of the infections produced by the different cell
lines varied markedly. Three animals that received B cells and
one that received CD8
1
T cells showed no clinical evidence of
infection. In a further five animals a few parasites were de-
tected in the regional lymph node for 1 to 4 days and there was
transient fever. Only in two animals (B944 and C165), both of
which received CD4
1
T cells, was there a moderately severe
reaction, with prolonged fever and high levels of parasitized
cells (.10%) in the regional lymph nodes; however, these two
animals cleared the infection on days 13 and 18 (Table 1).
There was therefore no consistent correlation between the
phenotype of the infected cells and the severity of the infec-
tions that they produced.
All of the animals were challenged in two groups, along with
susceptible controls, with a lethal dose of T. parva (Muguga)
sporozoites at 4 and 12 weeks after the initial infection. All
except two of the animals developed mild or inapparent reac-
TABLE 1. Reactions of cattle inoculated with autologous T. parva-infected cell lines of different phenotypes
Animal
No. of
cells
Source of
cell line
Phenotype of cell line
a
Parasitosis in regional lymph node
CD4 CD8 WC1 Ig Onset (day) Level
b
Duration (days)
B944 5 3 10
4
PBMC 1222 7 111 6
C166 1 3 10
5
PBMC 1622 5 111 11
C428 1 3 10
5
PBMC 1122 6 1 4
C244 1 3 10
5
PBMC 1622 6 1 1
C190 1 3 10
5
PBMC 2122 8 1 2
C197 1 3 10
5
PBMC 2122 nd
D673 1 3 10
5
WC1
1
cells 2612 6 1 2
D647 1 3 10
5
sIg
1
cells 2222 nd
C672 1 3 10
5
sIg
1
cells 2222 nd
C193 1 3 10
5
sIg
1
cells 2222 nd
D648 1 3 10
5
sIg
1
cells 2222 9 1 2
a
1, all cells in the culture were positive for the marker; 6, only a portion of the cells were positive; 2, none of the cells were positive.
b
111, 11, and 1, levels of parasitosis of .10%, 1 to 10%, and ,1%, respectively; nd, not detectable.
558 MORRISON ET AL. INFECT.IMMUN.

tions, in some instances with a few parasites being detected for
up to 4 days in the regional lymph node (data not shown),
indicating that they had developed immunity. Two of the ani-
mals that initially showed no reaction following inoculation
with infected B-cell lines (C193 and C672) developed severe
infections with prolonged fever and high levels of parasitosis in
regional and contralateral lymph nodes. One of these animals
(C193) died on day 21, whereas the other cleared the parasites
on day 22 and recovered.
Reactions of animals inoculated with purified cell popula-
tions infected in vitro with T. parva. The relatively mild infec-
tions produced by cloned parasitized cell lines were considered
to reflect selection of infected cells and/or the parasite, during
the period of culture required to clone, expand, and charac-
terize the parasitized cell lines. To overcome this problem, four
experiments were carried out in which various cell populations
purified from individual animals by cell sorting were infected
with sporozoites and inoculated into the donor animal after
culture for 48 h. Doses ranging from 3 3 10
4
to 3 3 10
5
cells
were administered in the different experiments. The resultant
infections were compared with that produced by a similar dose
of whole PBMC subjected to the same staining and sorting
procedures and cultured under the same conditions. In these
experiments, all animals that continued to exhibit fever and
high levels of parasites in regional and other lymph nodes were
killed on day 16.
The cell types that the animals received and the reactions of
the individual animals following infection are presented in
Table 2. In two experiments supernatants from infected whole
PBMC were collected after 48 h of culture and inoculated into
a susceptible animal. Both animals (D812 and D836) remained
negative for parasites, showed no clinical signs of infection,
and were fully susceptible to subsequent challenge with a lethal
dose of sporozoites. On the basis of these findings and similar
previous results (22), it was concluded that there were no
surviving extracellular sporozoites in the 48-h cultures.
In all four experiments, animals that received infected
PBMC developed severe reactions, with fever commencing
between days 6 and 9 and parasites initially being detected in
the regional lymph node on day 6 or 7 and attaining persis-
tently high levels in both the regional and contralateral lymph
nodes. In the first two experiments, individual animals were
inoculated with purified autologous T cells (CD2
1
) or B cells
infected 48 h previously with sporozoites. In each instance, the
animal that received T cells exhibited severe infection compa-
rable to that observed in animals inoculated with infected
PBMC. By contrast, the two animals that received infected B
cells (D822 and D826) developed mild self-limiting infections,
with parasites detected only in the regional lymph nodes. Par-
asitized cells were detectable for 3 and 5 days, reaching peak
levels of 0.2 and 5%, respectively; only one of these animals
exhibited fever.
In the third and fourth experiments (Table 2), infections
produced after inoculation of animals with purified CD4
1
and
after inoculation with CD8
1
T cells were compared. In each
experiment, both T-cell subsets produced severe infections
similar to that induced by whole PBMC. In the fourth exper-
iment, an additional animal (E312) was included, and it re-
ceived 2 3 10
6
infected purified B cells, i.e., 10 times the
number of cells given to animals infected with PBMC, CD4
1
cells, or CD8
1
cells. Despite the higher dose of cells, this
animal was able to control the infection. It developed fever and
had detectable parasitized cells for 9 and 8 days, respectively,
and had cleared the parasites by day 15. However, the level of
parasitosis in the regional lymph node did not exceed 1% and
parasites were detectable for only 1 day in the contralateral
lymph node.
Given the previous findings that B cells and T cells can be
infected at similar frequencies with T. parva (1), the results of
these experiments indicate that infected B cells and T cells, or
the parasites therein, differ in their capacity to replicate and
cause disease in vivo.
All three animals that recovered from infection following
inoculation with infected B cells were resistant to infection
when challenged with a lethal dose of T. parva (Muguga)
sporozoites 6 weeks later. Two of the animals exhibited fever
for 1 day, and a few parasites were detected in the regional
lymph node of only one animal (B312) for 3 days (data not
TABLE 2. Reactions of cattle inoculated with different cell populations 48 h after infection in vitro with T. parva sporozoites
Expt Animal
Cell
population
a
No. of
cells
Fever
Parasitosis
Local node Opposite node
Onset (day) Duration (days) Onset (day) Level
b
Duration (days) Onset (day) Level
b
Duration (days)
1 E107 PBMC 2 3 10
5
7 .97111 .99111 .7
D825 CD2
1
2 3 10
5
7 .96111 .99111 .7
D822 sIg
1
2 3 10
5
9 1 3
D836 Supt
2 D811 PBMC 3 3 10
5
6 .10 6 111 .10 9 111 .7
E110 CD2
1
3 3 10
5
6 .10 6 111 .10 8 111 .8
D826 sIg
1
3 3 10
5
64611 5
D812 Supt
3 F1 PBMC 3 3 10
4
9 .76111 .10 9 111 .7
E313 CD4
1
3 3 10
4
7 .96111 .10 9 111 .7
E332 CD8
1
3 3 10
4
6 .10 6 111 .10 9 111 .7
4 F22 PBMC 2 3 10
5
9 .76111 .10 9 111 .7
F10 CD4
1
2 3 10
5
7 .95111 .11 8 111 .8
F16 CD8
1
2 3 10
5
8 .86111 .10 9 111 .7
E312 sIg
1
2 3 10
6
94711 8101 1
a
Supt, supernatant from infected PBMC after 48 h of culture.
b
111, 11, and 1, levels of parasitosis of .10%, 1 to 10%, and ,1%, respectively. All animals that underwent severe reactions were killed 16 days after infection.
VOL. 64, 1996 CELL TYPES INFECTED BY T. PARVA 559

shown). Challenge control animals developed sustained fever
and high levels of parasitosis.
Phenotypes of parasitized cells in animals infected with
defined cell populations. In six of the animals used in experi-
ments 3 and 4 (Table 2), cells aspirated by a needle biopsy
technique from lymph nodes on day 11 of infection were
stained with a single T-cell marker and fractionated into pos-
itive and negative populations for detection of parasites. Cells
from one animal that received PBMC (F1) and two animals
that received CD4
1
T cells (E313 and F10) were sorted into
CD4
1
and CD4
2
populations, while cells from two animals
that received CD8
1
cells (E332 and F16) were sorted into
CD8
1
and CD8
2
populations. Cells obtained from the animal
inoculated with 2 3 10
6
purified B cells (E312) were sorted
into CD2
1
and CD2
2
populations. The results of these anal-
yses are shown in Table 3.
In the animal infected with whole PBMC, parasitized cells
were found within both the CD4
1
and CD4
2
populations,
although a majority were CD4
1
. By contrast, in animals that
received infected CD4
1
or CD8
1
cells, all of the detectable
parasitized cells were within the respective CD4
1
or CD8
1
populations. Thus, as far as could be detected, the parasite was
still confined to the T-cell type originally infected. However, in
the cells from the animal that received 2 3 10
6
infected B cells,
parasitized cells were detected in the CD2
1
but not in the
CD2
2
population, indicating that most, if not all, of the patent
infection on day 11 was attributable to T cells.
Infected B cells are prominent in early cultures of infected
PBMC. Despite the previous finding that purified B cells and T
cells become infected and establish cell lines at similar fre-
quencies in vitro, the majority of cell lines established from
mixed populations of cells have a T-cell phenotype (1, 21),
suggesting that development of the parasite in B cells or
growth of parasitized B cells might be impaired in the presence
of other cell types. However, in none of the previous studies
were infected cell lines derived from PBMC analyzed during
the early stages of establishment to determine whether in-
fected B cells were present. Whole PBMC were therefore
infected in vitro with saturating levels of sporozoites and the
level of infection and surface expression of Ig and the T-cell
markers CD2, CD4, and CD8 were examined after different
periods of culture. The results of the surface phenotype anal-
yses for four cell lines are shown in Table 4. In this experiment,
transformation of the cultures was apparent after 4 to 5 days,
and by day 7 more than 85% of the cells were parasitized
lymphoblasts. On day 7, large numbers of surface Ig
1
(sIg
1
)
cells were found in all four cultures (ranging from 22 to 68%),
and in three of the four cultures sIg
1
cells outnumbered the
cells expressing T-cell markers. However, by day 14, the num-
bers of sIg
1
cells had markedly declined (,8%), and by days
44 and 50 the cells in all four cultures were essentially all
positive for T-cell markers. At this time, one of the cultures
was predominantly CD4
2
CD8
1
whereas cells in the other
three cultures were all CD4
1
, with a variable proportion (16 to
79%) also expressing CD8. These results indicate that in mixed
cell populations there is a substantial early growth of parasit-
ized B cells, which are eventually overgrown by T cells. There
is also an apparent selection of one T-cell subset or another in
individual cultures.
DISCUSSION
Primary infections of cattle with T. parva are characterized
by rapid multiplication of parasitized lymphoblasts which be-
come disseminated throughout the lymphoid system and are
responsible for much of the pathology associated with infection
(16, 23). Thus, the disease is considered an inevitable conse-
quence of the capacity of the parasite to induce transformation
of host lymphocytes such that parasite replication outpaces
protective cellular immune responses. The findings of the
present study modify this view of the pathogenesis and dem-
onstrate that the outcome of infection is influenced by the host
cell type infected by the parasite.
Studies of the cell tropism of T. parva in vitro have demon-
strated that B cells and CD4
1
or CD8
1
a/b T cells and g/d T
cells are all susceptible to infection and transformation by the
parasite at more or less similar frequencies (1). While most
established lines of parasitized B cells lose expression of sur-
TABLE 3. Phenotypes of parasitized cells in lymph nodes of cattle
inoculated with purified cell populations infected with T. parva
Animal
No. and type of
infecting cells
Lymph node
tested
a
Sorted
population
%
Parasitosis
F1 3 3 10
4
PBMC Regional CD4
1
78.0
CD4
2
4.0
E313 3 3 10
4
CD4
1
Regional CD4
1
70.0
CD4
2
,0.1
E332 3 3 10
4
CD8
1
Regional CD8
1
69.0
CD8
2
,0.1
F10 2 3 10
5
CD4
1
Contralateral CD4
1
12.5
CD4
2
,0.1
F16 2 3 10
5
CD8
1
Contralateral CD8
1
1.0
CD8
2
,0.1
E312 2 3 10
6
sIg
1
Regional CD2
1
5.0
CD2
2
,0.1
a
All lymph nodes were tested on day 11 of infection.
TABLE 4. Phenotypes of T. parva-infected cell lines at different
times after establishment by infection of PBMC
in vitro with sporozoites
Donor
animal
Cell surface
marker
a
% of cells positive for marker on
day after infection
b
7144450
E98 IgM 52 5 1 2
CD2 9849696
CD4 5549191
CD8 3601716
E182 IgM 22 2 NT 0
CD2 39 82 NT 98
CD4 29 54 NT 92
CD8 6 54 NT 79
E223 IgM 51 2 0 0
CD2 23 88 97 98
CD4 16 67 98 98
CD8 6397378
E293 IgM 68 8 1 1
CD2 8689594
CD4 5 49 2 7
CD8 2439492
a
The CD8-specific MAb used in this experiment (IL-A51) detects both the
homodimeric (a/a) and heterodimeric (a/b) forms of CD8.
b
NT, not tested.
560 MORRISON ET AL. INFECT.IMMUN.

Citations
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Journal ArticleDOI
TL;DR: The presence of the parasite in the host-cell cytoplasm modulates the state of activation of a number of signal transduction pathways, including nuclear factor-kappa B, which appear to be essential for the survival of Theileria-transformed T cells.
Abstract: Theileria parva and T. annulata provide intriguing models for the study of parasite-host interactions. Both parasites possess the unique property of being able to transform the cells they infect; T. parva transforms T and B cells, whereas T. annulata affects B cells and monocytes/macrophages. Parasitized cells do not require antigenic stimulation or exogenous growth factors and acquire the ability to proliferate continuously. In vivo, parasitized cells undergo clonal expansion and infiltrate both lymphoid and non-lymphoid tissues of the infected host. Theileria-induced transformation is entirely reversible and is accompanied by the expression of a wide range of different lymphokines and cytokines, some of which may contribute to proliferation or may enhance spread and survival of the parasitized cell in the host. The presence of the parasite in the host-cell cytoplasm modulates the state of activation of a number of signal transduction pathways. This, in turn, leads to the activation of transcription factors, including nuclear factor-kappa B, which appear to be essential for the survival of Theileria-transformed T cells.

149 citations


Book ChapterDOI
TL;DR: Analysis of the cytotoxic T-cell response has revealed evidence of BoLA haplotype dominance plus competition between parasite epitopes, but further vaccine development will probably require identification of a range of other antigens, especially from the schizont stage.
Abstract: There are three economically important bovine Theileria species: Theileria annulata, which causes tropical theileriosis and occurs across north Africa and most of central Asia; Theileria parva, which causes East Coast fever and is found in East and Central Africa; and Theileria sergenti, which is predominantly a problem in Japan and Korea. Theileria annulata preferentially infects macrophages in vivo. It is controlled largely by means of live, attenuated vaccines, which are produced by prolonged tissue culture of the schizont-infected cells. The immunity induced in animals, which have either recovered from an infection or have been vaccinated (with an attenuated vaccine), is broad, solid and cell mediated. It is considered that the main effector cells are cytostatic macrophages that produce nitric oxide. Subsidiary roles for bovine leucocyte antigen (BoLA)-restricted, transiently appearing, cytotoxic T cells, and possibly also natural killer (NK) cells, have been identified. Cytokines such as tumour necrosis factor alpha (TNF-alpha) may have important roles, particularly in the induction of pathology. Matrix metalloproteinases have been implicated in the metastatic behaviour of schizont-infected cells. The nature of the protective schizont target antigens remains unknown. Attempts to develop a subunit vaccine have focused upon a sporozoite antigen (SPAG-1) and a merozoite antigen (Tams1). Both SPAG-1 and Tams1 have given partial protection using different delivery systems and adjuvants, but further vaccine development will probably require identification of a range of other antigens, especially from the schizont stage. Theileria parva has a tropism for T cells. Vaccination is currently by the 'infection and treatment' method, which involves challenging with a controlled dose of sporozoite stabilate and the simultaneous administration of long-acting tetracyclines. The immunity thus induced is mediated by BoLA-restricted cytotoxic T cells, which recognize polymorphic schizont antigens. These antigens have not been characterized at the molecular level. However, the polymorphic nature of the target antigens underlies the fact that the immunity is very strain specific--a situation that distinguishes T. parva from T. annulata. Interestingly, it is not possible to produce an attenuated vaccine to T. parva, as T. parva requires up to two orders of magnitude more schizonts in order to achieve transfer to the new host. A suggested reason for this is that the macrophage targets of T. annulata are phagocytes and thus the schizont has a natural, efficient route of entry whilst the preferred host of T. parva is the non-phagocytic T cell. Analysis of the cytotoxic T-cell response has revealed evidence of BoLA haplotype dominance plus competition between parasite epitopes. Subunit vaccination using a recombinant sporozoite antigen (p67) has proved very promising, with levels of protection of the order of 70% being achieved. A proportion of the protected calves exhibits complete sterile immunity. Interestingly, the basis for this immunity is not clear, since there is no correlation between the titre of antibodies that inhibit sporozoite penetration of lymphocytes and protection. Similarly, there is no significant T-cell response that distinguishes the protected and susceptible animals. These data are very encouraging, but other components, particularly those derived from the schizont, need to be identified and characterized. The mild Theileria species of Japan and Korea (termed T. sergenti in the literature) cause fever and severe chronic anaemia. The schizont stage of the life cycle is very rare and the host cell type is not known. The pathology is associated with chronic piroplasm infection. Immunity can be induced by immunizing with crude piroplasm extracts. Serological analysis of immune sera reveals that the immunodominant antigen is a polypeptide of 30-33 kDa, which corresponds to the protective T. annulata polypeptide Tams1. (ABSTRACT T

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TL;DR: It is proposed that comparisons between cancer biology and host-Theileria interactions can reveal chemotherapeutic targets against Theileria-induced pathogenesis based on cancer treatment approaches.
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TL;DR: The Muguga cocktail ITM vaccine, which provides broad-spectrum immunity to ECF, is now a registered product in three countries in eastern Africa and effort is directed at improving and scaling up the production process to make this vaccine more widely available on a commercial basis in the region.
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TL;DR: It is suggested that the initial sporozoite binding event triggers the mobilization of intrasporozoite Ca2+ and the activation of both kinase and G-protein associated signalling processes in the parasite, which regulate the invasive capacity of the sporozoites.
Abstract: Theileria are important tick-transmitted protozoan parasites that infect wild bovidae and domestic animals throughout much of the world. Much of our understanding of Theileria sporozoite invasion of bovine cells is based on work on T. parva , the causative agent of East Coast fever in cattle throughout east, central and southern Africa. Sporozoite entry involves a defined series of sequential but separable steps that differ in important details from the invasion process in other apicomplexans such as Plasmodium and Toxoplasma . While the morphological features of invasion are fairly well documented, the detailed biology of the individual steps is only now becoming clear. This review summarizes much of this recent work on the biology of sporozoite entry. In particular, recent studies on the role of Ca 2+ and cell activation processes in sporozoite entry suggest that the initial sporozoite binding event triggers the mobilization of intrasporozoite Ca 2+ and the activation of both kinase and G-protein associated sigualling processes in the parasite. These processes in turn regulate the invasive capacity of the sporozoite although the identity of these parasite molecules and how they contribute to the invasion process remain to be determined.

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References
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Journal Article
Abstract: We report the identification of a subset of bovine T cells by two monoclonal antibodies (mAb), IL-A11 and IL-A12, and some functional analyses of these cells. Both mAb precipitate two polypeptides, called BoT4, with apparent molecular mass of 52 and 55 kilodaltons. The epitopes recognized by these two mAb are different, however. BoT4 is found on approximately 70% of thymocytes and 30% of peripheral blood mononuclear cells (PBM), is not expressed by monocytes or B cells, and is found on cells in the T-dependent areas of lymph nodes. BoT4+ lymphocytes purified by a fluorescence-activated cell sorter proliferate in response to mitogenic and alloantigenic stimulation without addition of exogenous growth factors, whereas BoT4- lymphocytes do not. Monoclonal antibodies IL-A11 and IL-A12 have no effect on mitogen- (PHA and Con A) or alloantigen-induced proliferation of PBM. Monoclonal antibody IL-A12 has no inhibitory effect on the cytolytic activity of bulk populations of alloreactive T lymphocytes, and most of the cytolytic activity generated in mixed leukocyte culture is ascribable to the BoT4- population. Using cloned alloantigen-specific lymphocytes, we found that the ability of BoT4+ clones to proliferate to alloantigenic stimuli without exogenous growth factors is a characteristic of some clones, as is susceptibility to inhibition of proliferation by mAb IL-A12. These results implicate the BoT4 molecule in antigen recognition but indicate that the requirement for BoT4 is variable among clones. Our findings, together with those in the companion paper, which provides evidence that BoT4+ lymphocytes are class II restricted, indicate that BoT4+ lymphocytes are the bovine equivalent of Leu3/T4+ lymphocytes of humans and analogous lymphocytes of other species.

204 citations


Journal ArticleDOI
TL;DR: Results indicate that the IL‐A29 and CC15 antibodies define a unique population of CD4−CD8−, γ/δ T cells.
Abstract: In this study, two monoclonal antibodies, IL-A29 and CC15, are described that identify a novel bovine cell surface marker of 215/300 kDa. The antibodies reacted with a discrete population of resting lymphocytes in peripheral blood which, in young animals, constituted about 25% of the mononuclear cells. Thymus, lymph nodes and spleen contained less than 5% positive cells. These cells were negative for surface Ig, a monocyte/granulocyte marker, and the T lymphocyte antigens CD2, CD6, CD4 and CD8. Immunohistological analyses revealed the presence of IL-A29/CC15-positive lymphocytes in the thymic medulla, in the outer cortex of lymph nodes, in the marginal zones of the spleen, in the dermal and epidermal layers of the skin and in the lamina propria of the gut. The IL-A29/CC15+ cells in unfractionated blood mononuclear cells responded in autologous and allogeneic mixed lymphocyte cultures, and when purified they responded to concanavalin A in the presence of recombinant interleukin 2. These observations suggested this population of cells belonged to the T cell lineage. In order to unambiguously define their lineage, cDNA clones encoding bovine T cell receptor (TcR) and CD3 proteins were isolated. Northern blot analyses of IL-A29/CC15+ cell populations and of established cell lines of various lineages demonstrated that they expressed TcR delta and CD3 gamma, delta and epsilon mRNA: TcR alpha was not expressed, whereas only a truncated form of TcR beta mRNA was present. These results indicate that the IL-A29 and CC15 antibodies define a unique population of CD4-CD8-, gamma/delta T cells.

202 citations


Journal ArticleDOI
14 Sep 1973-Nature
TL;DR: Techniques recently developed in this laboratory can be used to infect cattle reproducibly with East Coast fever with infective particles of Theileria parva collected from the tick vector, Rhipicephalus appendiculatus.
Abstract: Infection and Transformation of Bovine Lymphoid Cells in vitro by Infective Particles of Theileria parva

200 citations


"Pathogenicity of Theileria parva is..." refers background in this paper

  • ...This characteristic of parasitized cells allows them to be propagated in vitro as continuously growing cell lines; such lines can be established with cells taken from infected cattle (20) or by infection of lymphocytes in vitro with sporozoites (4)....

    [...]


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TL;DR: Fractionation of effector cells generated in an allogeneic mixed leucocyte culture (MLC) demonstrated that cytotoxic cells specific for class I major histocompatibility complex (MHC) antigens reside within the BoT8+ population.
Abstract: Monoclonal antibody (mAb) IL-A17 characterizes a subset of bovine T lymphocytes. IL-A17 recognizes a 34,000-35,000 MW doublet, designated BoT8, which is expressed on the surface of approximately 20% of peripheral blood mononuclear leucocytes (PBM), a subpopulation of lymphocytes in T-dependent areas of lymph nodes and spleen, and about 70% of thymocytes. This molecule is not expressed on B lymphocytes, monocytes/macrophages or granulocytes. Double labelling of PBM showed that the BoT8+ population is distinct from the T lymphocyte subset expressing BoT4. BoT8+ lymphocytes purified with a fluorescence-activated cell sorter (FACS) proliferated poorly in response to mitogenic and alloantigenic stimulation in the absence of exogenous growth factors. IL-A17 had no inhibitory effect on proliferation of PBM to mitogens (Con A and PHA) or alloantigens and no measurable effect on the in vitro generation of cytolytic effector cells. However, in some experiments IL-A17 was found to block partially allospecific cytolytic function mediated by bulk effector cell populations when included in 51Cr-release assays. Fractionation of effector cells generated in an allogeneic mixed leucocyte culture (MLC) demonstrated that cytotoxic cells specific for class I major histocompatibility complex (MHC) antigens reside within the BoT8+ population. Based on these data, and information reported elsewhere on alloreactive bovine T-cell clones, BoT8 is considered to be analogous to CD8 in humans and equivalent molecules in other species.

139 citations


Journal ArticleDOI
15 Aug 1964-Nature
TL;DR: Within the first 30 days maintenance on pyridoxine-deficient diet, with or without daily oral treatment with L-penicillamine, does not causo either weight loss or mortality in CF! mice.
Abstract: weight did occur, albeit at a slower rate than with complete diet, and there was no mortality. The a.ddition of L-penicilla.mino to the drinking water of the group maintained on pyridoxine-deficient diet retarded their normal weight gain still further within the first 30 days, but thero was no actual weight loss. It was only during the second 30 days of oral L-penicillamine therapy with pyridoxinedeficient diet that a 15 per cent loss in body-weight and 20 per cent mortality occurred. This was attributed to the reinforcing action of L-penicillamine on the vitamin deficiency induced by pyridoxine-deficient diet. Baseline data demonstrate tha.t within the first 30 days maintenance on pyridoxine-deficient diet, with or without daily oral treatment with L-penicillamine, does not causo either weight loss or mortality in CF! mice.

137 citations


"Pathogenicity of Theileria parva is..." refers background in this paper

  • ...An association with the mitotic spindle enables the parasite to divide synchronously with the lymphocyte (14)....

    [...]