Raphal Guatteo, DMV, Msc, PhD, Dipl ECBHM

UMR 1300 Oniris-INRA BioEpAR

Infection
60% asymptomatic
Incubation :
2 3 weeks
2% of infected
persons
Chronic
disease
Endocarditis,
40% Acute Disease
- Flu-like syndrom
- Hepatitis
- Pneumonia
- Meningitis
4% need for hospitalization
Pregnancy disorders
- Abortions (repeated)
- Foetal death
- Premature delivery
Adapted from Dupuis, 1985
Brianon (05) 1996
Banon (04) 1987
goats
Brasparts (29)
2002 goats
Nordheim(67) 2005
goats
Chamonix
2002
Ewes
Cholet
2009
Veal
Montoison (26)
2001 goats
Brianon (05) 1996
Near slaughterhouse Ewes (Eastern day)
Endemic situation around Etang de Berre (13)
Annual occurrence +++ > mean occurence
Outdoor Lambing (wind +++)
Source : Anses (E. Rousset)
Florac
2008
Sheep
foetal
serum
Mainly Limited outbreaks but
Occurrence similar to
Toxoplasmosis (human
aboprtion)
Sometimes outbreak +++
Q fever: a Zoonosis with outbreaks
2009
2357 Human cases
60% asymptomatic
20% pneumonia leading to hospitalization
Goats +++ Goats +++
Control actions
BTM Monitoring
Mandatory vaccination (Phase 1 vaccine)
Culling (stamping out) pregnant
Goats and/or Ewes
Recent Outbreak in Hungary
Not Rare
Ruminants as main source for
human infection
Q fever: Prevalence in Ruminants ?
Species
Prevalence at
animal level
Median [Min-Max]
Prevalence at
herd level
Median [Min-Max]
Prevalence at
within herd level
Median [Min-Max]
Cattle 20% [0-100%] 40% [0-100%] 20% [0-60%]
Sheep 11% [0-100%] 25% [0-100%] 30% [0-70%]
Goat 17% [0-100%] 26% [0-100%] 25% [20-85%]
Guatteo et al., 2011
Most frequently Serological study (Risk Assessment ?)
Need for harmonization for Epi study
Human Health Issue
Dairy Industry Issue
Reduction of Coxiella burnetii shedding
Inhalation
Raw
Milk ?
Inhalation +++
Q fever: Human Health Issue.only?
7
Reduction of Coxiella burnetii shedding
Limit the zoonotic risk
Satisfaction of dairy industry
Inhalation
Q fever in Ruminants : Clinical Impact ?
Reproduction: Myth or Reality ?
Adapted from Human pathogenesis
Supported by field observations (?)
APSW
Abortions and/or then retained placenta, metritis Abortions and/or then retained placenta, metritis
Premature delivery
Stillbirth
Weak Calf syndrome
Other signs: metritis, retained placenta,
fertility disorders ? respiratory disorders ?
Invasion and Localisation in the placenta
Establishment of
infection
Active infection
Clearance
Immediate clearance of
infection
Latent infection
Limited spread Spread to the foetus
Latent infection Active infection Widepread Localized
Most Likely outcome:
normal offspring
Possible outcomes : APSW syndrome
Or normal offspring
Blood
spread
Transplacental
spread
Liver
Amniotic
fluid
Bowel Lung
Localized or disseminated
infection
3 complementary approaches
Experimental reproduction
Healthy animals
Challenged
Observed Observed
Case/control study
Retrospective or cohort study
Difference in prevalence of reproductive disorders depending on
the exposure
Intervention study
Implementation of control actions, routine diagnosis
Comparison before vs after, with vs without
Experimental Reproduction
Pro Cons
Impact-interaction with other
Precise effect of THIS infection
Controlled conditions
Different infectious dose
Impact-interaction with other
pathogens
Costly, Limited number of animals
Inoculation often different from natural
route
Impact of included animals (eg
pregnant or not?)
2 studies: one in goat / one in cattle
In goats (Arricau-Bouvery et al., 2005)
0 -42 -21 84 100 - 157
6 weeks
mating
Vac ph
I et 2
0 -42 -21 84 100 - 157
6 weeks
post
abortions
necropsy

Vac ph
I et 2
Inoculation
CbC1 10
4
Not vaccinated group 12 goats
Vaccine Phase II group 15 goats
Vaccine Phase I group 16 goats
Abortions
Delivery
In goats (Arricau-Bouvery et al., 2005)
Abortions +++
Consistent with abortion storm's reported under field conditions
Other signs not investigated
In cattle (Plommet et al., 1973)
12 heifers
8-11 months old
Intradermal inoculation Intradermal inoculation
18 months follow-up
In cattle (Plommet et al., 1973)
Abortions +, Subfertility +
Consistent with signs reported under field conditions
Lack of detection for other pathogens
Experimental Reproduction: finally
Demonstration Not known
Abortions +++
Other reproductive disorders
(retained placenta, metritis)
Subfertility +/- Impact of pregnancy stage on
susceptibility
Case control study
Pro Cons
Retrospective study (cost-effective)
Deal with available data
Precise definition of case & control
Concomitant Identification of risk
factors
May lead to identify slight effect (size
of the dataset)
Precise definition of case & control
If cross sectional survey: association
but no causality
May lead to identify statistical
effect (size of the dataset) not
supported by biology
Case control study in cattle : 7 studies
Critical review
Exhaustive analysis in the proceeding
Number of animals : > 300 vs < 300: vs o
Control group (ie not exposed): vs o
Sensitive detection method (ELISA >>FC): vs o
Reference Sample Analysis Reproductive
disorders
collected/studied
Main results about
prevalence
Reliability
score
To et al., 207 dairy cows Serum immunofluorescence Metritis, infertility Serum testing: 3.9% PCR
Example of poor design
To et al.,
1998
207 dairy cows
who suffered from
previous
reproductive
disorders (93
infertility and 114
metritis and
mastitis
Serum immunofluorescence
(IF), serum PCR, milk PCR,
milk bacteriology.
Metritis, infertility Serum testing: 3.9% PCR
+, milk testing: 24.6%
PCR + (Coxiella burnetii
has been isolated in
24.6% of PCR + milk
samples)
58.9%IF + (phase I)
60.4%IF + (phase II)
O O
Reference Sample Analysis Reproductive disorders
collected/studied
Main results about
prevalence
Reliability
score
Literak and 1397 dairy cows Complement fixation First artificial insemination Correlation between
Example of intermediate quality study
Literak and
Kroupa,
1998
1397 dairy cows
at dry period (14
herds)
Complement fixation
test (phase II antigen)
First artificial insemination
(AI1) success rate, final
conception rate, calving-
AI1 interval, calving
interval, average number
of AI until pregnancy
Correlation between
infection
seroprevalence and
reproductive
performance not
statistically significant
o
Reference Sample Analysis Reproductive
disorders
collected/studied
Main results about
prevalence
Reliability
score
Lopez-Gatius
et al., 2011
3 herds reproductive
disorders (811 dairy cows)
(pregnancy rate: 23%,
Serum ELISA Retained placentas,
stillbirths, abortions,
embryonic resorptions
Correlation between
seropositivity and
retained placenta
Example of well designed study
(pregnancy rate: 23%,
abortion rate: 21%, BTM
testing: RT-PCR +, 2
control herds (2371 cows)
(pregnancy rate: 38%,
abortion rate: 11%, BTM
testing:RT-PCR
embryonic resorptions retained placenta
(almost twice more of
retained placentas in
seropositive cows).
No correlation between
prevalence and
abortion rate after 90
days but low incidence
of these events

Reference Sample Analysis Reproductive disorders
collected/studied
Main results about
prevalence
Reliability
score
Ordronneau,
2012
2825 cows from
affected herds (with
Serological test (Cb01
strain) performed in
Abortions and retained
placentas during the 6
Abortion risk increased 2.5
times in seropositive
Example of well designed study
abortions) all females older than
24 months
following months animals (in comparison
with seronegative ones)
Risk of retention increased
1.5 times in seropositive
animals (in comparison
with seronegative ones)

Case control study: finally
Demonstration Not known Demonstration Not known
Abortions +++
Retained placenta ++
Subfertility +/-
Metritis
Retained placenta itself or retained
placenta following abortion
Co-infections ?
Intervention study
Field investigation in case of disease: real life
Clinical trial (vaccine, antibiotic)
Pro Cons
Concomitant assessment of the
effectiveness of control strategies
(including placebo)
May lead to hypothesis about the
pathogenesis
Comparison of clinically and not
clinically affected animals/herds
Inclusion criteria crucial (under or
overestimate the treatment efficacy) :
diagnosis !!
Measurement of the treatment
efficacy itself of the natural course of
the infection (ie time)
Costly, limited number of situations
(including co-infections)
Field investigation of abortions
925 abortions investigated using PCR (Guatteo et al., 2014)
Coxiella burnetii
Chlamydophila spp
Listeria monocytogenes
Salmonella spp.
Campylobacter fetus (fetus fetus et fetus venerealis)
Anaplasma phagocytophilum
BHV4
Leptospira interrogans
Pathogens n %
Coxiella burnetii 160 17,3%
Anaplasma phagocytophilum 57 6,2%
Salmonella spp 38 4,1%
Listeria monocytogenes 16 1,7%
Leptospira pathognes 16 1,7%
BHV4 15 1,6%
Chlamydophila spp 10 1,1%
Campylobacter fetus fetus et venerealis 5 0,5%
Total 317 34,3%
40
50
60
N
u
m
b
e
r

o
f

s
a
m
p
l
e
s
0
10
20
30
<10 [10-15[ [15-20[ [20-25[ [25-30[ [30-35[ [35-40[ [40-45]
N
u
m
b
e
r

o
f

s
a
m
p
l
e
s
Distribution of Ct values
15
20
25
30
35
N
u
m
b
e
r

o
f

s
a
m
p
l
e
s
[40-45]
[35-40[
[30-35[
0
5
10
N
u
m
b
e
r

o
f

s
a
m
p
l
e
s
Cb load
[25-30[
[20-25[
[15-20[
[10-15[
<10
When combining PCR pos and Ct value: Q fever : around 10% of
repeated abortions
25
30
35
40
N
u
m
b
e
r

o
f

s
a
m
p
l
e
s
Placenta Mucus vaginal Avorton Estomac
0
5
10
15
20
<10 [10-15[ [15-20[ [20-25[ [25-30[ [30-35[ [35-40[ [40-45]
N
u
m
b
e
r

o
f

s
a
m
p
l
e
s
Ct values
Months N
Q fever
n %
Jan 116 27 23,3%
Feb 90 21 23,3%
Mar 92 14 15,2%
April 84 15 17,9%
May 41 11 26,8% May 41 11 26,8%
June 48 12 25,0%
July 90 15 16,7%
Aug 111 22 19,8%
Sept 107 6 5,6%
Oct 49 7 14,3%
Nov 32 3 9,4%
Dec 36 4 11,1%
Sample n
Fivre Q
n %
Aborted calf 30 5 16,7%
Stomach content 36 5 13,9%
Placenta 480 89 18,5%
Mucus 353 55 15,6% Mucus 353 55 15,6%
Pregnancy Pregnancy Pregnancy Pregnancy stage ( stage ( stage ( stage (month month month month) )) ) Q Q Q Q e!er e!er e!er e!er "re!a#ence "re!a#ence "re!a#ence "re!a#ence
$0 $0 $0 $0% %% %1& 1& 1& 1& 0'
&1 &1 &1 &1% %% %2& 2& 2& 2& 20'
&2 &2 &2 &2% %% %3& 3& 3& 3& 6'
&3 &3 &3 &3% %% %4& 4& 4& 4& 14'
&4 &4 &4 &4% %% %5& 5& 5& 5& 1(' &4 &4 &4 &4% %% %5& 5& 5& 5& 1('
&5 &5 &5 &5% %% %6& 6& 6& 6& 16'
&6 &6 &6 &6% %% %)& )& )& )& 15'
&) &) &) &)% %% %*& *& *& *& 1)'
&* &* &* &*% %% %(& (& (& (& 2)'
Days after abortion Weeks after abortion
0 1 2 3 1 2 3 4 5 6 7 8
+ + + + + + + + - - - -
+ + + + + + + + - - - -
+ + + + + - - - - - -
+ + + + + + + + + - -
+ + + + + + + + + - - -
Goats
+ + + + + + + + + - - -
+ + + + + + + - - - - -
+ + + + + + - - - - -
+ + + + + + - - - - -
+ + + + + + - - - - -
+ + + + + + + - - - -
+ + + + + + + - - - - -
+ + + + + + + - - - - -
Arricau-Bouvery et al. 2005
Cattle
Cow
D0 D14 D21 D28
PCR result
1 + - - +
2 + + + +
3 + - - -
4 + - - -
5 + - - -
6 + + + -
7 + + - -
8 + + - + 8 + + - +
9 + + + +
10 + + - -
11 + - - -
12 + - - -
13 + + - -
14 + - - -
15 + - - -
16 + - - -
17 + - - -
18 + - - -
19 + - - -
20 + - - -
21 + - - -
22 + - - -
23 + + - -
24 + - - -
Daprs Guatteo et al. 2012
Studies dealing with vaccine efficacy
Both in goats and cattle
In goats
mainly focused on abortions mainly focused on abortions
experimental conditions
In cattle
abortions and fertility
3 studies under field conditions
$ $$ $Arr+cau Arr+cau Arr+cau Arr+cau% %% %,ou!ery ,ou!ery ,ou!ery ,ou!ery et a#.- 2005- .acc+ne& et a#.- 2005- .acc+ne& et a#.- 2005- .acc+ne& et a#.- 2005- .acc+ne&
Vaccination scheme
Vaccine not challenged phase I phase II not vaccinated
Number of goats 27 16 15 12
Duration of gestation (days)
ab
150 (+1.8) 153 (+3) 134 (+15) 141 (+8)
Abortion (%)
b
15 6 87 75 Abortion (%)
b
15 6 87 75
Number of kids per goat 1.9 (+0.88) 1.5 (+0.52) 1.67 (+0.62) 1.75 (+0.87)
Percentage of goats with contaminated
placenta (%)
b

ND 37.5 93.3 100

Very strong prevention effect in susceptible animals
Limit : in small ruminants: very high within-herd
seroprevalence at abortion time
[Arricau-Bouvery et al., 2005, Vaccine]
Vaginal shedding
10
12
14
16
n
u
m
b
e
r

o
f

s
h
e
d
d
e
r

g
o
a
t
s
NV
Mean duration of shedding
NV 22 days
0
2
4
6
8
10
DO D1 D2 D3 W1 W2 W3 W4 W5 W6 W7 W8
time after kiddings
n
u
m
b
e
r

o
f

s
h
e
d
d
e
r

g
o
a
t
s
NV
Ph II
Ph I
NV 22 days
Ph II 16 days
Ph I 1,5 day
D-17 D-2 D0 D21 D90
D180
D270 D360 CALVING
M, VM, F, BS x 2
M, VM, F
M, VM, F M, VM, F M, VM, F M, VM, F
Randomization
[Guatteo et al., 2008, Vaccine]
Treatment 1
Vaccination
or placebo
Treatment 2
Vaccination
or placebo
Determination
of
initial status
Detection of shedding
Shedding follow up among initially susceptible animals
Survival analysis method (Cox regression model)
Description of bacterium load when shedding occurred
6 clinically infected herds
336 animals
/n+t+a# status /n+t+a# status /n+t+a# status /n+t+a# status
Status
Non
pregnant
Pregnant Total
Cows
S 30 62 92
$0uatteo et a#.- 200*- .acc+ne& $0uatteo et a#.- 200*- .acc+ne& $0uatteo et a#.- 200*- .acc+ne& $0uatteo et a#.- 200*- .acc+ne&
40
Cows
NS 52 105 157
Heifers
S 30 53 83
NS 1 3 4
Total 114 222 336
0.75
1.00
0.75
1.00
0.75
1.00
Efficacy of the
vaccine
$0uatteo et a#.- 200*- .acc+ne& $0uatteo et a#.- 200*- .acc+ne& $0uatteo et a#.- 200*- .acc+ne& $0uatteo et a#.- 200*- .acc+ne&
0.00
0.25
0.50
FUP
0 100 200 300 400 500
Vaccinated when non-pregnant
Vaccinated when pregnant
Placebo
Censored animals are marked with circles
0.00
0.25
0.50
FUP
0 100 200 300 400 500
0.00
0.25
0.50
FUP
0 100 200 300 400 500
Vaccinated when non-pregnant
Vaccinated when pregnant
Placebo
Censored animals are marked with circles
When vaccinated non pregnant,
the risk of becoming shedder was 5 times lower
Vaccination and side effects on reproduction
In France, April of 2005 March of 2006
115.562 vaccine phase I (Coxevac) doses: cattle/goat/sheep
- 0.08 % of local reactions
42
- 0.08 % of local reactions
- 0.003 % of transitory hyperthermia
- 0.002 % of mortality
- 0.012 % of abortion
- 0.04 % of other (loss of appetite, decrease of milk
production)
Vaccination and side effects on reproduction
Both goats [Arricau-Bouvery et al., 2005] and cattle [Guatteo et al., 2008] studies
Any notable induration
Any increase of Body Temperature
DG
at D35
Calving between
D0 and D35
Abortion Early calving
DG+
at D35
DG+
at D0
Farm
DG
at D35
Calving between
D0 and D35
Abortion Early calving
DG+
at D35
DG+
at D0
Farm
3
0
0
3
0
0
0
at D35
9
2
2
0
5
0
0
D0 and D35
1 1 109 123 total
0 0 12 14 6
0 0 18 20 5
1 (PCR-) 0 23 27 4
0 0 19 24 3
0 0 17 17 2
0 1 20 21 1
at D35 at D0
3
0
0
3
0
0
0
at D35
9
2
2
0
5
0
0
D0 and D35
1 1 109 123 total
0 0 12 14 6
0 0 18 20 5
1 (PCR-) 0 23 27 4
0 0 19 24 3
0 0 17 17 2
0 1 20 21 1
at D35 at D0
[Guatteo et al., 2008]
Common treatment for all herds: VACCINATION of Nulliparous
120 Dairy Herds (repeated abortions related to Q fever)
R
A
N
D
O
M
Vaccination
Antibiotics
None
Vaccination
Antibiotics
In herds with Antibiotics : cows assigned at random:
1 or 2 administrations at drying off
1 administration at calving
Phase 1 Vaccine (Coxevac, CEVA Sant Animale),
Long acting Oxytetracycline (20mg/kg, TENALINE LA, CEVA)
M
1 or 2 administrations at drying off and calving
$ $$ $1aure# 1aure# 1aure# 1aure# et a#.- 2013& et a#.- 2013& et a#.- 2013& et a#.- 2013&
Comparison of abortions and retained placenta
occurrence (-6 : +12 m) between groups
Description of return to service rate between groups
Herd Animal
Environment
Vaginal Swabs (22 herds)
At calving
BTM or Primiparous mix
Monthly or quarterly
Dust, bedding
Effectiveness to
reduce/prevent Cb shedding
at individual level
Indicator of Cb circulation at
herd level
Environmental Cb Load
Effectiveness of bedding removal
10
11
12
13
14
15
16
1)
A
b
o
r
t
i
o
n

r
a
t
e

%
Within herd abortion rate (median) according to treatment group
3.*
5
3.3
4.5
2.3
2.4
2.1
2.2
0
1
2
3
4
5
6
)
*
(
10
.acc+n .acc 2 A1,Q A1,Q 3ontro#
A
b
o
r
t
i
o
n

r
a
t
e

%
4eore +nc#us+on
ater +nc#us+on
Clinical signs Results
Abortions
(6 months before
abortions vs 12
Vaccination: OR : 0,694 [0.453-1.06] (P=0.09)
Seropositivity (p<0.05) :
Prevention of reproductive disorders
abortions vs 12
months after)
Seropositivity (p<0.05) :
OR : 2,547 [1.709-3.795]
Retained placenta
Seropositivity (p<0.05) : OR: 1.526 [1.061-2.195]
No effect of vaccination
Confirmation of implication of Coxiella in reproductive disorders
Trend to reduce abortions in cows
Vaccination in
nulliparous
Vaccination in
Cows
ATB at calving
Return to
service
(18-26 d)
ns ns ns
Improvement of fertility
(18-26 d)
Return to
service
(27-90 d)
OR : 0,538 [0.301-
0.963] (p<0.05
ns
OR: 0.759 [0.597-
0.965] (P<0.05)
Confirmation of interest of vaccination in nulliparous
Limited effect of antibiotics
Variable N
Cb load > 10000 vs. ]0-100] b/mL
OR P
Vaccination 0.03
Prevention of shedding
None
76 1
After service
73 0.29
Before service
13 0.15
Vaccination (cows and heifers)
Reduction of Cb load shed when shedding occurred
Reduction of the transmission rate
74 herds, 354 BTM samples, 5254 dairy cows
Vaccination
covering rate
0-20%
Vaccination
covering rate
20-80%
Vaccination
covering rate
>80%
Reduction of shedding at herd level
Favourable
pattern in BTM
(reduction of Cb
load over time)
0,29 0,17 1
Whole herd vaccination (>80%) : Strong and fast reduction of Cb
shedding at herd level
Reduction of shedding in environment (bedding)
80%
100%
Interest of vaccination to reduce Cb load
Interest of bedding renewal to decrease infectious pressure
0%
20%
40%
60%
D0 D90 D180 D270 D360 D450 D540
Negative
Low Cb load
Moderate Cb load
High Cb load
High ++ Cb
load
2 dairy herds
PCR positive on bulk tank milk
Determination serological status all > 12 months
Random allocation (sero status)
301 control and 310 vaccine (Coxevac) at 170 and
[Lopez-Gatius et al., 2014]
301 control and 310 vaccine (Coxevac) at 170 and
190 days of pregnancy
Follow up of return to service, conception rate at first
service, calving to conception interval
Results
25 % of cows were seropositive (effectiveness of the
vaccine +++ in seronegative animals)
[Lopez-Gatius et al., 2014]
Vaccine
group
Control
group
P value
group group
Conception
rate at first
service
42% 30% 0,04
Number of AI
per pregnancy 1.9 +/-0.1 2.3 +/- 0.2 0,03
CCI 92 106 0,02
Intervention study: finally
Demonstration Not known
Abortions +++
Retained placenta ++
Return to service ++
Metritis
Retained placenta itself or retained
placenta following abortion
Co-infections ?
To summarize
Species ASPW
Complex
Endometritis Fertility
disorders
Retained
placenta Complex disorders placenta
Cattle + ? + +
Goat + ? ? +
Sheep + ? ? ?
Outcome Vaccination
Abortions Occurrence
Impaired fertility Return to service Impaired fertility Return to service
Coxiella burnetii
shedding
Reduction of level of shedding in cows at calving
Prevention of shedding in nulliparous at calving
Whole vaccination : Fast/strong reduction of shedding
at cow and herd level
Safety No adverse reaction
Including in pregnant animals
Acknowledgements
All the cows, farmers and vets included in the studies ....
In memory of Christophe Manteca