Veterinary Immunology and Immunopathology 107 (2005) 217–233

www.elsevier.com/locate/vetimm

Gamma interferon-producing CD4 T-cells correlate with

resistance to Mycoplasma mycoides subsp. mycoides
S.C. infection in cattle
L. Dedieu a,*, V. Balcer-Rodrigues a, A. Yaya b, B. Hamadou b,
O. Cisse c, M. Diallo c, M. Niang c
a
CIRAD, Animal Health Programme, TA30/G, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France
b
Laboratoire National Veterinaire, BP 503 Garoua, Cameroon
c
Laboratoire Central Veterinaire, PO Box 2295, Route de Koulikoro Km8, Bamako, Mali
Received 13 April 2004; received in revised form 27 April 2005; accepted 28 April 2005

Abstract

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC), is one of the
most significant cattle disease in Africa. The control measures, which led to eradication from numerous countries are not feasible
in Africa where the only prophylaxis relies on vaccination. However, the attenuated vaccines, used up to now in Africa, are of
low efficiency. The development of an improved vaccine is, therefore, a necessity.
The purpose of this study was to compare some immunological parameters in MmmSC-infected cattle (endobronchial versus
natural in-contact infection) and assess the response in correlation with the clinical outcome (death versus recovery).
Characterization of the immune parameters elicited in recovered animals, known to be refractory to new infection, will be
an important step towards development of new vaccines against CBPP.
A significant outcome of this study was the demonstration that all MmmSC-infected cattle developed a MmmSC-specific
cell-mediated immune response.
A kinetic analysis of the MmmSC responsiveness showed that the main difference between endobronchially- and in-contact
infected animals was the delay before the onset of the MmmSC-specific immune response. The first MmmSC-responding PBMC
sample was selected from each animal for cell phenotyping. The phenotypic analysis of this early MmmSC-induced response
revealed the predominant contribution of the CD4 T-cells in all animals whereas IFNg was only constantly produced in
recovered animals.
Evolution of this early MmmSC-specific immune response was then followed by a kinetic analysis of the MmmSC-induced
CD4 T-cell response and IFNg released. The results demonstrated that in recovered animals, the MmmSC-specific CD4 Th1-like
T-cell response was maintained until slaughtering whereas in animals with acute disease, progression of CBPP was associated
with a decreased ability of the PBMC to produce IFNg.

Abbreviations: CBPP, contagious bovine pleuropneumonia; MmmSC, Mycoplasma mycoides subsp. mycoides Small Colony; MdFI,
median of fluorescence intensity
* Corresponding author. Tel.: +33 4 67593816; fax: +33 4 67593798.
E-mail address: laurence.dedieu@cirad.fr (L. Dedieu).

0165-2427/$ – see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2005.04.011
218 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233

The results led to the identification of immune parameters, which correlate with protection against CBPP and to a relevant
strategy for the development of improved vaccines against this disease.
# 2005 Elsevier B.V. All rights reserved.

Keywords: Contagious bovine pleuropneumonia; Mycoplasma mycoides subsp. mycoides S.C.; Vaccine; Cell-mediated immunity; Gamma
interferon

1. Introduction cedure was widely used in Europe and South Africa

until the policy of eradication by stamping-out was
Contagious bovine pleuropneumonia (CBPP), adopted (Turner, 1959). Since then, various types of
caused by Mycoplasma mycoides subsp. mycoides vaccines have been tested (Provost, 1974; Provost
biotype Small Colony (MmmSC), is one of the most et al., 1987; Turner, 1959). Nowadays CBPP
serious cattle diseases in Africa. The principal prophylaxis in Africa relies on the use of live vaccines
pathological mechanism is a strong inflammatory based on the attenuated T1 strain. However, the
reaction, usually restricted to the lungs, leading to efficacy of the T1 vaccines is low and they induce only
death by respiratory distress due to lung consolidation short-term protection, making annual vaccination
in 15–30% of cases. In most cases, however, CBPP necessary to achieve a sufficient level of protection
evolves into a chronic form, followed by recovery (Thiaucourt et al., 2000). As a consequence, the
(Provost et al., 1987; F.A.O., 2003). development of an improved vaccine is a prerequisite
In recent years, CBPP has emerged from areas where for the eradication of CBPP in Africa. This objective is
it has been persisting in endemic form to reinvade zones based on Willems’s experiments and on the long-term
from which it had previously been eradicated (Bots- immunity to reinfection observed in CBPP-recovered
wana, Tanzania, Rwanda. . .). In addition to these cattle, demonstrating their ability to mount a
newly-infected areas, even the endemic zones are protective immune response against MmmSC (Provost
experiencing an upsurge in incidence. The disease is et al., 1987).
responsible for heavy economic losses due to mortality, The basis of immune protection against MmmSC
loss of weight, reduced working ability or fertility. infection has so far remained unknown. Previous
Furthermore, CBPP is the only bacterial disease immunological studies have primarily examined
included in List A of the Office International des serological responses, but with contradictory results
Epizooties (OIE), and infected countries are excluded concerning their role in protection (Lloyd, 1967;
from international trade. It is therefore considered as the Masiga et al., 1975). However, while a role for
most important threat to the cattle industry in Africa. antibody-dependent mechanisms cannot be ruled out,
Although CBPP was widespread until the middle of cell-mediated immunity is likely to be involved in
the 19th century, its distribution has been considerably protection (Roberts et al., 1973; Roberts and Windsor,
reduced through the successful application of control 1974; Tulasne et al., 1996).
measures. It has been completely eradicated from The role of the cellular immune response in the
numerous countries such as Australia, the USA and pathogenesis and control of MmmSC infection has
many European countries through a combination of a never been analysed, nor have the T-cell subsets
stamping-out policy, control of cattle movement and involved been identified. However, an understanding
quarantine. However, such measures are impracticable of the protective and immunopathological mechan-
in Africa, where the only realistic prophylaxis is isms will determine the most appropriate and efficient
through vaccination. vaccine strategy against CBPP.
The history of CBPP vaccination dates back to This study explores a number of immunological
1852, when Willems established that subcutaneous parameters in MmmSC-infected cattle and assesses the
inoculation of CBPP-infective lymph in the thick immune response in correlation with the clinical
connective tissue protected cattle against contact outcome of the infection (fatal acute infection versus
challenge (Willems, 1852). This immunisation pro- recovery). Characterisation of the immune parameters
 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233 219

of CBPP-recovered animals, known to be refractory to Etheridge, 1983). Thirteen naive peul zebus were put
reinfection, will be an important step towards the in-contact with 12 N’dama cattle infected with MmmSC
development of new efficient vaccines against CBPP. during a field outbreak. The animals, 3- to 6-year-old,
were housed in close contact for 7 months.
Two control animals were included in each experi-
2. Materials and methods ment and were kept separate from the infected animals.
Blood samples were collected repeatedly from each
2.1. Strains and growth conditions animal, starting prior to infection until the slaughter-
ing. Temperature and clinical signs were recorded
MmmSC strains were cultured in Hayflick medium daily. Animals with respiratory distress were slaugh-
containing 15% horse serum, 7% yeast extract, 0.4 g/l tered as well as all animals at the end of the experiment
sodium pyruvate and 0.1 g/l glucose. Mycoplasmas and a post-mortem (PM) analysis was realised.
were collected in log phase growth by centrifugation at A serological follow-up of the MmmSC infection
10,000  g for 20 min and washed two times in PBS was carried out by complement fixation test (CFT),
buffer. The number of bacteria was determined by recommended by the OIE as the standard serological
microtitration in liquid media. Aliquots were then kept test for CBPP diagnosis (Dedieu et al., 1996).
at 20 8C until used. The Afade strain was used for Three endobronchially-infected cattle from experi-
experimental endobronchial infection. For in vitro ments 1 and 6 naturally-infected ones from experi-
antigen stimulation assays, mycoplasmas were heat- ment 2 were selected for the immunological study. The
killed by 1-h incubation at 60 8C. The MmmSC total selected zebus from experiment 2 were 4-year-old
proteins were titrated by the bicinchoninic acid (C4, C12 and C14) and 5-year-old (C9, C11 and C13).
method (Smith et al., 1985).
2.3. PBMC preparation
2.2. Experimental infections
PBMC were isolated by differential centrifugation
Nine cattle were selected from two experimental over lymphocyte separation medium (Eurobio, Les
infections for this immunological study. Both experi- Ulis, France) and washed in calcium- and magnesium-
ments involved animals taken from CBPP-free areas free Hanks balanced salt solution (Eurobio, Les Ulis,
and never vaccinated against CBPP. All animals were France) to which 100 U/ml penicillin, 100 mg/ml
selected for their negative status for foot and mouth streptomycin and 250 ng/ml amphotericin B (Sigma,
disease, tuberculosis and brucellosis and were St. Quentin, France) had been added. The PBMC were
dewormed before use. Protocols were designed and stored frozen at 2.5  107 cells/ml in foetal calf serum
performed in accordance with the French national supplemented with 10% DMSO (Sigma, St. Quentin,
legislation for animal experimentation. France) and shipped to France under liquid nitrogen.
In experiment 1, conducted in Cameroon (National
Veterinary Laboratory, Garoua), six 4-year-old Bororo 2.4. Lymphoproliferation assays
zebus were anaesthetised by I.M. injection of xylacin
(Rompun-Bayer) and positioned in right lateral PBMC were rapidly thawed by transfer to a 37 8C
recumbency. A bronchoscope was then gently water bath and resuspended to 2.5  106/ml in RPMI-
introduced up to the bronchus and 5  108 CFU of 1640 culture medium (Eurobio, Les Ulis, France)
the Afade strain mixed with 1.5% of agar were then supplemented with 2mM L-glutamine (Eurobio, Les
delivered in the right lung followed by 50 ml of sterile Ulis, France), 5  10 5 M 2-mercaptoethanol, 100 U/
culture medium (Poumarat et al., 1989; Abdo et al., ml penicillin G, 100 mg/ml streptomycin, 0.25 mg/ml
1998). The animals were kept for 5 months. amphotericin B, 200 ng/ml gentamycin (Sigma, St.
Experiment 2 was conducted in Mali (Central Quentin, France) and 10% fetal calf serum (Eurobio,
Veterinary Laboratory, Bamako), using a larger number Les Ulis, France). One hundred microlitres of PBMC
of animals because of the difficulty of transmitting were distributed into each well of a 96-well microtitre
CBPP (Gourlay and Howard, 1982; Lloyd and plate. The PBMC were incubated with whole heat-
220 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233

killed MmmSC (5 mg/ml), either with Concanavalin A differences between MmmSC-stimulated and non-
(Sigma, St. Quentin, France) (2.5 mg/ml) as a positive stimulated samples. However, for comparative analy-
control, or in RPMI media as a negative control. All sis, only cell percentages higher than 2% (i.e. 100
tests were performed in triplicate. Cultures were events out of 5000 events acquired) were considered.
incubated for 5 days at 37 8C with 5% CO2. T-cell activation was monitored for each T-cell
subset using gate settings. The data are expressed in
2.5. Search for a superantigen-like molecule in figures as the percentage of CD25-expressing cells
MmmSC strains within the CD4+, CD8+ and WC1+ T-cell gates,
respectively. The relative activation of the CD4 T-cells
PBMC samples from five naive cattle were was evaluated by measuring the median of fluores-
analysed by lymphoproliferation assay as described cence intensity (MdFI) of the gated CD25+CD4
above, except that various concentrations of whole T-cells (Overton, 1997; Hope et al., 2003). All data
heat-killed MmmSC were used, ranging from 100 ng were expressed as the mean and standard deviation
to 5 mg of protein/ml. of the cell percentage differences between MmmSC-
stimulated and non-stimulated samples.
2.6. Cell phenotyping
2.7. IFNg quantification
The surface phenotype of PBMC was analysed by
indirect immunofluorescence staining using specific Lymphoproliferation assay supernatants were col-
mouse monoclonal antibodies (MAb) to the following lected on day 5 to quantify gamma interferon (IFNg)
bovine leukocyte antigens: CD2 (IL-A26), CD4 (IL- production using a commercially available enzyme-
A12), CD8 (IL-A105), WC1 (gd T-cells, CC15), MHC linked immunosorbent assay (Bovigam g interferon
class II (J11), interleukin-2 receptor CD25 (IL-A111) test, BioCore, Omaha, NE) in accordance with the
(Baldwin et al., 1988; MacHugh et al., 1993; Naessens manufacturer’s instructions. Positive (PBMC incu-
et al., 1997). The MAbs were obtained from the bated with ConA) and negative (PBMC incubated in
International Livestock Research Institute (ILRI, media) controls were included in each assay. The
Nairobi, Kenya). MAb DU2-104 reacting with B-cells optical density (OD) values were the means of
was kindly provided by W. Hein (Basel Institute for triplicate assays. Results were deemed positive when
Immunology, Basel, Switzerland (Mukwedeya et al., the OD of MmmSC-stimulated cell supernatant minus
1996). negative control was 0.2.
Cells were incubated for 1 h at 4 8C with various
combinations of three MAbs, washed, stained with 2.8. Statistical analysis
conjugated goat anti-mouse immunoglobulins for 1 h
at 4 8C and washed. The conjugates were fluorescein According to the small number of animals, a non-
isothiocyanate (FITC), phycoerythrin (PE) or tri- parametric test (distribution-free), the Mann-Whitney
colour (TC)-labelled anti-isotype antibodies (TEBU U-test, was used for the statistical analysis. p-Values
S.A, Le Perray, France). Control was performed with less than 0.05 were considered significant.
normal mouse serum.
A three-colour flow cytometric (FACS) analysis
was then performed by the fluorescence-activated cell 3. Results
sorter (FACscan; Becton Dickinson, San Jose, CA)
and the CellQuest software, acquiring 5000 events. 3.1. Experimental infections
Light-scattering profiles (forward and side scatter)
permitted the exclusion of dead cells and debris from Table 1 presents the results of the animals selected
data analysis and the assessment of blastogenesis. for the study from both experimental infections.
Results were determined as the percentage of Clinical signs (temperature, cough, nasal discharge,
fluorescing cells and were expressed as the mean dyspnoea) were similar in both experiments, although
and standard deviation of the cell percentage more pronounced in experiment 1.
 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233 221

Table 1
Results of the experimental infections
Animals Clinical form Outcome Onset of Clinical signs PM analysis Date of
disease seroconversion (CFT)
Experiment 1
I1 Acute Death (3 wpi) 2 wpi ++++ H, A, EIS, PF 2 wpi
855 Chronic Recovery 2 wpi +++ S 2 wpi
001 Chronic Recovery 3 wpi +++ Resolved lesions 3 wpi
Experiment 2
C12 Acute Death (26 wpc) 10 wpc ++ H, EIS, PF 24 wpc
C14 Acute Death (19 wpc) 10 wpc ++ H, EIS, PF 19 wpc
C4 Chronic Recovery 9 wpc + Resolved lesions 13 wpc
C9 Chronic Recovery 11 wpc ++ A, S 13 wpc
C11 Chronic Recovery 10 wpc + A, S 10 wpc
C13 Chronic Recovery 12 wpc + Resolved lesions 21 wpc
PM, post-mortem; CFT, complement fixation test; wpi, weeks post infection; wpc, weeks post contact; H, hepatisation; A, adherence; EIS,
enlargement of interlobula septa; PF, pleural fluid; S, sequestrum.

Animals with acute disease, characterised by an onset of clinical signs, seroconversion was observed
accelerated evolution of the clinical symptoms leading 2–3 wpi in experiment 1 and varied from 10 to 24 wpc
to respiratory distress, were slaughtered. The PM in experiment 2.
analysis revealed typical characteristics of CBPP: only No correlation can be established between the onset
one lung affected, solid and marbled appearance of the of the disease, the CFT data and the different clinical
lung with areas of different colours (indicating forms of CBPP.
different stages of hepatisation evolving towards
necrosis) separated by a network of pale bands 3.2. Ex vivo systemic cellular immune response
(interlobular septa), large quantity of pleural fluid in analysis
the chest cavity with accumulation of fibrin leading
the lung and chest wall to stick together. An ex vivo phenotypic analysis of the PBMC taken
Recovered animals presented a regression of the from each animal during the course of the experiment
clinical signs. The PM examination showed only was performed. The kinetics of each cell population
adhesions, between lung lobes or lungs and chest wall, (B-cells, gd, CD4 and CD8 T-cells and monocytes)
and sequestra, i.e. areas of dead (necrotic) tissue and state of activation (CD25 and MHC class II
surrounded by a capsule of fibrous connective tissue, molecule expression) were monitored by flow
demonstrating the resorption or sequestration of the cytometry. However, no significant observation for
lung lesions. any cell subset can be correlated with the clinical
No clinical signs of CBPP or serological data were forms and outcome of the MmmSC infection (data not
observed for control animals. shown).
The main difference between experiments 1 and 2
was in the length of the incubation period. Endo- 3.3. MmmSC-specific cell-mediated immune
bronchially-infected animals developed the disease 2– response
3 weeks post infection (wpi) whereas the length of
incubation in naturally infected animals varied from 9 3.3.1. Preliminary assays with PBMC from naive
to 12 weeks post contact (wpc) for the selected animals
animals, and to 28 wpc if all animals initially included The presence of superantigen-like molecules in
in experiment 2 are considered (Niang et al., 2005). MmmSC was assessed before MmmSC-specific
For each animal, Table 1 also shows the date of immune responses were analysed. PBMC from five
seroconversion determined by CFT. Similarly to the naive animals were stimulated for 5 days with various
222 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233

concentrations, from 100 ng to 5 mg protein/ml, of 3.3.2. Screening of MmmSC-responding PBMC

heat-killed MmmSC. The results showed no MmmSC- samples
induced blastogenesis or cell activation, whatever the A preliminary screening was performed to select
MmmSC antigen concentration (data not shown). The for each animal the MmmSC-responding PBMC
absence of MmmSC-induced non-specific lymphocyte samples during the course of the infection.
activation enabled the MmmSC-specific immune Kinetic studies of MmmSC-induced blastogenesis
response in infected cattle to be studied. were carried out by incubating PBMC samples with
A concentration of 5 mg of protein/ml of heat- 5 mg/ml of whole heat-killed MmmSC for 5 days.
killed MmmSC was retained for the lymphoprolifera- Positive control based on ConA stimulation was
tion assays. The data obtained at this MmmSC systematically included to verify cell viability. For
concentration were used to define the cut-off point experiment 1 animals, kinetics was established using
to discriminate between positive and negative values PBMC taken at several time points until 20 wpi. In
for MmmSC-induced blastogenesis, percentage of experiment 2, because of the long incubation period,
CD25+CD4 T-cells and MdFI of this subset. The cut- samples were selected for each relevant period: before
off points, determined by the mean values plus two infection, clinical periods, time of seroconversion and
standard deviations of MmmSC-induced minus nega- during months 6 and 7, the final months before slaughter.
tive control, were 3, 5 and 20%, respectively, rounded Fig. 1 presents the kinetics of MmmSC-induced
to integers. blastogenesis for all animals. A MmmSC-induced

Fig. 1. kinetic analysis of the MmmSC-responsiveness of the PBMC samples. Stimulation and analysis were performed as described in Section
2. Values represent the difference between MmmSC-induced and medium control values. The star indicates the period of seroconversion as
measured by the CFT.
 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233 223

blastogenesis was observed in all infected animals, MmmSC. The phenotypes of the MmmSC-responding
revealing the presence of MmmSC-primed lymphocytes cells were then compared between endobronchially-
circulating in blood. The results showed that the onset infected and naturally-infected animals (Fig. 2).
of MmmSC responsiveness varied greatly according to Comparative analysis was also performed between
the mode of infection. MmmSC-primed lymphocytes animals with acute CBPP and recovered animals
were detected in blood as early as 2 weeks after (Fig. 3). Data are expressed as the mean and standard
endobronchial infection. In contrast, 11–28 weeks were deviation of the cell percentage differences between
required in the case of natural infection, although four MmmSC-stimulated and non-stimulated samples.
of the six animals demonstrated a positive response 13– Assessment of the CD25+ T-cells in each T-cell
15 wpc. None of the PBMC samples from the control subset was expressed as a percentage of the respective
animals presented MmmSC-induced blastogenesis gated T-cell subset.
above the 3% cut-off point. The phenotyping of MmmSC-induced cell activation
No significant correlation can be observed between showed marked similarities, whatever the route and
onset of disease, humoral response, MmmSC-specific outcome of the MmmSC infection. Figs. 2 and 3 show
systemic cell-mediated immune response and clinical that in vitro MmmSC recall induced activation of the
forms and outcome (death versus recovery) of infection. three T-cell subsets as measured by up-regulation of
Although 2 of the 3 endobronchially-infected animals CD25 expression, although with differences in magni-
developed the disease and a MmmSC-specific cellular tude, whereas no MmmSC-induced T-cell activation
and humoral response 2 wpi, animal 855 recovered was noted in the control animals. An MmmSC-induced
from the MmmSC infection whereas animal I1 died increase in the percentage of cells expressing the
from an acute form of the disease (Fig. 1, Table 1). activation marker CD25 was observed among each T-
Similarly, all naturally-infected animals developed the cell subset. The increase in CD4 T-cells expressing
disease almost 10 wpc. However, two of the five CD25 was always significant as the CD25+CD4 T-cell
animals presenting an early MmmSC-specific systemic subset was always greater than the cut-off point stated
cellular immune response 11–15 wpc died from the for FACS analysis of cell subsets (2% of total cells, see
infection (C12 and C14) and three recovered (C4, C9 Section 2). In contrast, the CD25+gd T-cell subset data
and C11). Moreover, the last animal (C13) charac- were non-significant (<2% of total cells) for one of the
terised by the most delayed MmmSC-specific cellular three endobronchially-infected samples and three of
response (28 wpc) also recovered. The only major the six naturally-infected samples. Similarly, the
difference noted in experiment 2 between recovering CD25+CD8 T-cell subset data were also non-significant
and dying animals was in the onset of the humoral and for one of the three endobronchially-infected samples
cellular responses: the humoral response, in the two and four of the six naturally-infected samples. However,
animals with acute CBPP, was detected late (8–10 although the increase in gd and CD8 T-cells expressing
weeks) after the cellular response, whereas in all CD25 after MmmSC stimulation was non-significant, it
recovered animals it was observed before or during the was always higher in infected cattle than in control
same week as the cellular response. animals.
As well as by percentage of CD25+CD4 T-cells, the
3.3.3. Phenotyping of MmmSC–responding quantitative assessment of MmmSC-induced CD4 T-
PBMC samples cell activation was measured by the relative fluores-
To investigate the cell types involved in the onset cence intensity of the gated CD25+CD4+T-cell subset,
of the MmmSC-specific cell-mediated response, the monitored by the median of fluorescence intensity
first PBMC sample with a significant percentage of (MdFI). The results showed that the MmmSC-induced
MmmSC-induced blastic cells from each MmmSC- increase in the MdFI of the gated CD25+CD4 T-cells
infected animal was selected for phenotyping. observed in the PBMC from all MmmSC-infected
Lymphocyte subset proliferation and activation, animals was significantly higher ( p < 0.05) than in
measured by the expression of the interleukin-2 those from non-infected animals.
receptor (CD25), was monitored for each PBMC Comparative analysis revealed that the percentage of
sample after 5 days of stimulation with heat-killed MmmSC-induced CD25+CD4 T-cells was not signifi-
224 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233

Fig. 2. Phenotypic characterisation of the MmmSC-responding cells: Comparative analysis between endobronchially (n = 3) and naturally-
infected animals (n = 6). Stimulation and analysis were performed as described in Section 2. Values are expressed as the mean and standard
deviation of the difference between MmmSC-induced and medium control values for all corresponding animals. The percentage of CD25+ T-
cells was expressed among each gated T-cell subset. The MdFI was calculated on the gated CD4+CD25+ T-cells.

cantly different ( p > 0.05) between endobronchially- acute disease and recovered animals. In contrast,
infected (28.83  7.29) and naturally-infected although MdFI values are shown in Fig. 3, the strong
(20.96  7.04) cattle (Fig. 2). However, comparison variation in these values between endobronchially-
of the MdFI of this subset showed that levels of and naturally-infected animals prevented comparison
fluorescence were significantly higher ( p < 0.05) in of this parameter between animals with acute CBPP
endobronchially-infected (200.5  41.72) than in and those recovering. The results showed that the
naturally-infected (42.52  27.34) animals. percentage of MmmSC-induced CD25+CD4 T-cells
Comparative analysis of the CD25+CD4 T-cell was significantly higher ( p < 0.05) in recovered
percentage was then performed between animals with (27.23  5.52) than in animals with acute CBPP
 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233 225

Fig. 3. Phenotypic characterisation of the MmmSC-responding cells. Comparative analysis between recovered (n = 6) and animals with acute
disease (n = 3). Stimulation and analysis were performed as described in Section 2. Values are expressed as the mean and standard deviation of
the difference between MmmSC-induced and medium control values for all corresponding animals. The percentage of CD25+ T-cells was
expressed among each gated T-cell subset. The MdFI was calculated on the gated CD4+CD25+ T-cells.

(16.24  6.39) (Fig. 3). However, because of the small were collected 5 days after MmmSC stimulation and
number of animals with acute disease, the analysis of a IFNg production was analysed by ELISA. IFNg
larger number of samples is required before this secretion was assessed by comparison with antigen-
observation can be validated. free control culture supernatant and results were
Figs. 2 and 3 also show that although an increase in deemed positive when the OD of MmmSC-stimulated
CD25+ cells was induced by MmmSC, no or very low cell supernatant minus negative control was 0.2.
MmmSC-induced expansion was observed for the The results showed that MmmSC always induced
CD4, gd or CD8 T-cell populations. Moreover, a slight significant ( p < 0.05) IFNg production (0.72  0.54
decrease in these populations after MmmSC stimula- OD units), although with strong variation, by PBMC
tion was noted in some experiments. from infected but recovered cattle, whatever the
infection protocol.
3.3.4. IFNg production In contrast, strong differences were observed in
To investigate whether MmmSC-reacting T-cells animals with acute disease according to the infection
were functionally active, PBMC culture supernatants protocol. In the two naturally-infected cattle, MmmSC
226 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233

stimulation did not induce IFNg (C14) or only slightly was not maintained. As described above, a CD4 T-cell
above the cut-off (0.24 OD units; C12), whereas strong response was in fact observed 2 wpi for I1, with strong
IFNg production (1.60 OD units) was noted in the IFNg production, 14 wpc for C12, with slight IFNg
endobronchially-infected animal (I1). This result may production and 11 wpc for C14, without IFNg
correlate with the higher relative level of MmmSC- production. The percentage and MdFI of CD25+CD4
induced CD4 T-cell activation observed with endo- T-cells then decreased below the cut-off points, except
bronchial infection and the lower level of CD4 T-cell for C12, but IFNg was no more detectable for all three
activation noted in naturally-infected cattle develop- animals.
ing an acute form of the disease. Fig. 4 also shows that the kinetics of the MmmSC-
Results from the control animals were all negative, induced CD4 T-cell activation for all naturally-
except for one sample (0.33 OD units) taken from an infected animals were consistent with those of the
animal at the end of the experiment, although without MmmSC-induced blastogenesis, indicating the invol-
MmmSC-induced T-cell activation. vement of the CD4 T-cells among the blastic cells. In
The study initially focused on the characterisation contrast, in the endobronchially-infected animal with
of the T-cell response involved at the onset of the acute disease, MmmSC-stimulation did not induce
immune response and demonstrated that CD4 T-cells CD4 T-cell activation of the PBMC taken 3 wpi,
were the main cells sensitised in vivo by MmmSC. The whereas MmmSC-induced blastogenesis was
CD4 T-cell response produced IFNg in all recovered observed. This result, and also the light-scattering
animals, whereas no conclusions can yet be drawn for profiles (data not shown), suggest that a different cell
cattle with acute disease. However, this analysis was population was involved in the MmmSC-induced
only performed on a single time point, corresponding blastogenesis. Confirmation of this observation
to the first MmmSC-responding cell sample. A kinetic requires the analysis of a larger number of samples.
analysis of the MmmSC-induced CD4 T-cell response
was then performed so as to have a clear under-
standing of the evolution of the immune response in 4. Discussion
recovered animals versus animals with acute disease.
The study examined the cell-mediated immune
3.4. Kinetic analysis of MmmSC-induced CD4 response induced in cattle by Mycoplasma mycoides
T-cell response subsp. Mycoides SC (MmmSC), the agent of con-
tagious bovine pleuropneumonia (CBPP).
A kinetic analysis of the MmmSC-induced CD4 The study focused on a comparative characterisa-
T-cell activation, based on the study of three parameters tion of the MmmSC-induced early immune parameters
(percentage of CD4 T-cells expressing CD25, MdFI of between animals with acute CBPP and recovered
the CD25+CD4 T-cell subset and IFNg production), animals. These were known to be refractory to new
was carried out. MmmSC infections and should be representative of a
Fig. 4 presents the results obtained for all animals protective immune mechanism.
although complete kinetics could not be established For immunological relevance, the approach was
for zebus I0 and 855 since some PBMC samples were based on natural infection (respiratory route) of the
either no longer available or non-responding to ConA natural host (cattle). However, experimental repro-
stimulation. duction of CBPP had often been described as difficult
Figure shows that all cattle recovering from the (Gourlay and Howard, 1982; Lloyd and Etheridge,
infection were characterised by a sustained MmmSC- 1983) and requires lengthy, thus costly, experimenta-
induced CD4 T-cell response with IFNg production up tion due to a long incubation period. This problem can
to month 7, when the animals were slaughtered. also be a limiting factor for vaccine efficacy testing.
However, as shown with animal C9, MmmSC-primed In addition to naturally-infected animals, the study
CD4 T-cells were not always circulating in blood. In therefore also included animals endobronchially-
contrast, in animals with acute disease, although an infected with MmmSC, as the latter may provide an
MmmSC-specific CD4 T-cell response was initiated, it attractive model to overcome these problems.
 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233 227

Fig. 4. Kinetic analysis of the MmmSC-induced CD4 T-cell activation and IFNg release. Stimulation and analysis were performed as described
in Section 2. Values represent the difference between MmmSC-induced and medium control values. (a) CD4 T-cell activation. Flow cytometry
analysis was done on gated CD4 T-cells and expressed as the percentage (% of cells) of CD4 T-cells expressing CD25 (&) and MdFI
(fluorescence Units) of the CD4CD25+ T-cell subset (&) (b) IFNg released measured by ELISA and expressed as OD Units .
228 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233

Fig. 4. (Continued ).

Our results confirmed that both infection protocols detected by CFT. The main difference between the two
induced similar clinical forms of CBPP in cattle, from experiments was in the length of the incubation period.
an acute form leading to death to chronic infection Two to three weeks were required for endobronchi-
leading to recovery. All MmmSC-infected animals ally-infected animals to develop the disease, whereas
developed an MmmSC-specific humoral response, strong variations in the length of the incubation period,
 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233 229

from 9 to 28 weeks, were observed in naturally- Previous studies had suggested the existence of an
infected animals, as previously described (Provost MmmSC-specific cellular immune response in CBPP
et al., 1987; F.A.O., 2003). but had not attempted further characterisation
A comparative analysis of the MmmSC-induced (Roberts et al., 1973; Roberts and Windsor, 1974;
immune response was then carried out between Tulasne et al., 1996).
endobronchially- and naturally-infected animals and In our study, the early MmmSC-induced immune
between animals with acute disease leading to death response was further characterised. For each animal,
and those with chronic infection evolving towards the first PBMC sample presenting MmmSC respon-
recovery. siveness was selected to phenotype the MmmSC-
As a preliminary approach, the study focused on responding lymphocytes. Our results demonstrated
the MmmSC-specific systemic immune response to that the early MmmSC-induced immune response was
characterise the early MmmSC-primed blood circulat- based on T-cell activation, although with some
ing T-cells. Local immunity might in fact have been difference in magnitude, whatever the infection
more relevant, however, bronchoalveolar lavages are protocol or clinical form of CBPP.
difficult to manage and standardise in cattle. The phenotypic analysis revealed the predominant
The absence of superantigen-like molecules in contribution of the CD4 T-cells in the MmmSC-specific
MmmSC was first verified since the ability of several immune response. CD4 T-cells were therefore the main
mycoplasma species to polyclonally activate B- or cells sensitised in vivo by MmmSC at the onset of the
T-cells has previously been described (Naot et al., infection for all MmmSC-infected animals.
1977; Sasaki et al., 1995; Simecka et al., 1993). The In vitro activation of gd and to a lesser extent of
study revealed that whereas heat-killed MmmSC had CD8 T-cells was also observed following MmmSC
no effect on naive PBMC in vitro, viable MmmSC- stimulation. Although non-significant in some ani-
induced a lymphocytopathic effect (Dedieu et al., in mals, in others an MmmSC-induced mean increase of
preparation). Accordingly, the in vitro lymphoproli- 30.8% (8.35%) in gd T-cells expressing CD25 and of
feration assays were performed using heat-killed 11.93% (3.35%) in CD8 T-cells bearing CD25 was
MmmSC. The characterisation of the mechanism noted. Studies based on depletion assays and
underlying the pathological cell interaction with inhibition of antigenic presentation is now underway
viable MmmSC is now underway. to assess the MmmSC-specific responsiveness of both
As a preliminary step, the MmmSC-induced cell cell types. Preliminary results suggested that gd T-cell
blastogenesis was monitored in PBMC during the activation is a CD4 T-cell dependent effect (personal
course of the infection to detect the early MmmSC- data), as already described for other pathologies
responding PBMC samples. This kinetic analysis (Hasvold et al., 2002; Okragly et al., 1996). It is likely
showed the appearance in blood of MmmSC-primed that the few activated CD8 T-cells were also
T-cells at different time points, in all animals. responding as bystanders to CD4 T-cell cytokines.
Endobronchial infection with MmmSC-induced an However, even if activated by CD4 T-cell cytokines,
earlier immune response (2–3 wpi) as the pathogen each of these T-cell types may play a role in the
was delivered directly into the host lungs. In contrast, the immune response.
delay varied from 11 to 28 weeks post contact in These data thus demonstrated a strong MmmSC-
naturally infected animals. These data show that induced activation of the CD4 T-cells at the onset of
although the onset of the MmmSC-specific immune the MmmSC-specific immune response and a less
response was determined by the infection protocol pronounced activation of the gd and CD8 T-cells,
(route but also MmmSC strain and dose), it was also through a ‘‘supposed’’ bystander effect.
largely dependent on the source of animals and The cytokines produced at the onset of the immune
individual variability, such as sensitivity to the infection. response were then investigated. As a preliminary
Accordingly, detection of the early MmmSC-primed step, the IFNg secreted by the MmmSC-reacting T-
circulating T-cells in naturally-infected animals cells was monitored. Study of Th2 cytokines in
required a large screening of the PBMC samples taken ruminants is in fact hampered by a lack of appropriate
regularly during the course of the experiment. ELISA tools. The results clearly showed that the
230 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233

MmmSC-induced T-cell response produced IFNg in tion in cattle where (1) the IFNg-response was
all recovered animals. Although all T-cell subsets are detected earlier than the antibody response and (2)
able to produce this cytokine, the dominant participa- the loss of putatively protective CD4 T-cells and the
tion of the CD4 T-cells in the MmmSC-specific decrease in IFNg production lead to lack of control of
response proved their involvement as a Th1-like type. the disease (Koets et al., 2002; Waters et al., 2003).
However, the Th cell paradigm is not as clear in cattle Failure to control the infection early in its course
and, although Th1 and Th2 T-cells can be found, Th0 allows bacterial multiplication and development of a
type T-cells secreting both types of cytokines may also major inflammatory reaction leading to severe
exist. The precise characterisation of the CD4 T-cells pathological consequences up to respiratory distress.
involved in such animals is now underway by A strong lung recruitment of the T and B-lymphocytes
intracellular cytokine labelling and flow cytometry upon disease progression may be responsible for the
analysis. No conclusions can be drawn for animals low peripheral MmmSC-responsiveness. Another
with acute disease because of the small number of hypothesis can be the unresponsiveness of the B-cells
animals tested and the strong variation in IFNg due to the impaired T-cell response. Indeed, it is likely
production observed between endobronchially- and that the strong inflammatory response overwhelms the
naturally-infected animals. This variation may be immune response. Several immunomodulating factors
explained by the higher level of CD4 T-cell activation (prostaglandins, cytokines) may be involved in a
noted in endobronchially-infected animals, probably switch of the T-cell immune reactivity leading to a
due to a higher amount of MmmSC antigens directly reduced ability to support B-cell expansion and
delivered into the lungs. antibody production. Comparative analysis of the
Although the functional role of the CD4 T-cells cytokines and immunoglobulin isotypes produced,
initially primed by MmmSC cannot yet be determined between animals with acute infection and recovered
in cattle with acute disease, the comparative kinetic animals, will allow a better understanding of the
analysis between animals with acute disease and those immunopathology of the disease.
recovering revealed strong differences occurring The MmmSC-specific CD4 Th1-like T-cell
during the course of the infection. In all recovered response observed, in contrast, in all recovered
animals, the MmmSC-specific CD4 Th1-like T-cell animals should correlate with a protective mechanism.
response was maintained until the date of slaughter. In Although, in the case of CBPP, resorption of the
contrast, progression of CBPP in all animals with infection is never complete, leading to the formation
acute CBPP was associated with a decreased ability of of sequestered lung lesions, the animals did in fact
the PBMC to produce IFNg, although CD4 T-cell recover and became protected against any new
activation was still present in one animal. A delayed MmmSC infection. Sequestra as well as granulomas
humoral response was also observed in the naturally develop as part of a protective host response to block
infected cattle with acute CBPP whereas antibodies the deleterious effects of the pathogens and prevent
occurred concurrently or preceded the cellular cell damage (Hasvold et al., 2002; Sandor et al., 2003).
response in all other animals. Although, the protective The recovered animals thus developed a protective
role of antibodies had never been demonstrated in this immune mechanism leading to control of the MmmSC
disease, they are likely to be involved. Therefore, infection, resolution of lung lesions or development of
animals with acute CBPP succumb because the sequestra and to the priming of appropriate CD4 Th1-
MmmSC infection escapes control by the immune like T-cells, probably responsible for a protective
system. Although, the small number of animals with anamnestic response against MmmSC.
acute disease hampered any conclusion, the present Induction of an MmmSC-specific CD4 T-cell
work indicates some potential key immunological response was predictable as extracellular pathogens
differences for further comparative studies between are likely to be processed for presentation in
animals with acute CBPP and those recovering from association with MHC class II molecules. Mycoplas-
the MmmSC infection. mas are in fact described as extracellular pathogens
A similar observation characterises the evolution of attached to the host cell surface, although few species
Mycobacterium avium subsp. paratuberculosis infec- have demonstrated in vitro mechanisms for entering
 L. Dedieu et al. / Veterinary Immunology and Immunopathology 107 (2005) 217–233 231

non-phagocytic cells (Rottem, 2002). However, data since neutrophil recruitment has been observed in
are lacking to confirm the inability of MmmSC to enter CBPP lung lesions (Rodriguez et al., 1996; Ferronha,
and survive in any type of host cell. personal communication). However, it is likely that
In contrast, the role of the CD8 T-cells in the MmmSC- the immune response induced by MmmSC is partly
specific immune response remains to be characterised. responsible for the lung damage observed in CBPP, as
Indeed, CD8 T-cell specific activation is restricted to already described for other mycoplasma-induced
MHC class I antigenic presentation induced by pneumonias (Cartner et al., 1998; Vanden Bush and
intracellularly produced heterologous antigens. Rosenbuch, 2003). Therefore, assessing the contribu-
The physiological role of gd T-cells are still tion of the immune system to CBPP pathology would
controversial and many hypotheses have been not only improve our understanding of the disease but
proposed. However, evidence is accumulating on might also give valuable information on the appro-
their contribution to protection against several priate vaccine strategy.
infections (Collins et al., 1998; Ferrick et al., 1996; This work is the first report on cell-mediated
Kronenberg, 1994). Nevertheless, their specificity has immune responses induced in cattle by the CBPP
been demonstrated only for a restricted number of agent. Our results favour the idea that susceptibility to
pathogens, suggesting that these cells can also be MmmSC infection is associated with a switch of the
indirectly activated by the cytokine milieu or by self- immune response to an impaired response, allowing
derived molecules (Daubenberger et al., 1999; bacterial dissemination and disease progression. On
Kronenberg, 1994; Okragly et al., 1996). The gd T- the other hand, recovery appeared to be linked with the
cells form a much larger proportion of the peripheral development of an efficient immune response mainly
blood cells in ruminants than in rodents and primates, based on IFNg-secreting CD4 T-cells. Accordingly, T-
although the population decreases with age. This study cell recognition of MmmSC antigens inducing IFNg
showed that bovine gd T-cells from MmmSC-infected release is of major importance in the development of
animals responded to in vitro stimulation with the protection against CBPP in cattle. We would therefore
whole pathogen whereas no activation was obtained in predict that vaccines priming CD4 T-cells to produce
naive PBMC. Their contribution to protection in cattle IFNg upon exposure to infection would induce
can therefore not be excluded and their specific role in protective immunity.
protective immunity against MmmSC will be analysed Studies are now underway both to further
further. characterise the MmmSC-specific immune response
CD4 T-cells should contribute to protection and the immunopathological aspects of the disease
through the release of cytokines such as IFNg. The and to identify MmmSC protective antigens.
principal function of IFN-g in vivo is macrophage
activation, with increased expression of MHC classes I
and II molecules, leading to enhancement not only of
Acknowledgment
the phagocytic capacity but also of cytokine secretion
and killing ability. Gamma IFN also promotes This work was supported by an European
expression of MHC class II molecules on endothelial
Commission research grant (ICA4-CT-2000-30015).
cells and a variety of epithelial cells. Accordingly,
The authors thank Garth Evans for his assistance with
IFN-g may increase the host cell capacity for
the English.
membrane-bound MmmSC capture, processing and
antigenic presentation. Furthermore, a Th1 response is
also characterised by a bias of the antibody response
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