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Evaluation of two commercial assays for the detection of antibodies
E. Vretou, F. Radouani, E. Psarrou, I. Kritikos, E. Xylouri, O. Mangana
To cite this version:
E. Vretou, F. Radouani, E. Psarrou, I. Kritikos, E. Xylouri, et al.. Evaluation of two commercial assays for the detection of antibodies. Veterinary Microbiology, Elsevier, 2007, 123 (1-3), pp.153.
�10.1016/j.vetmic.2007.02.023�. �hal-00532217�
Accepted Manuscript
Title: Evaluation of two commercial assays for the detection of Chlamydophila abortus antibodies
Authors: E. Vretou, F. Radouani, E. Psarrou, I. Kritikos, E.
Xylouri, O. Mangana
PII: S0378-1135(07)00103-4
DOI: doi:10.1016/j.vetmic.2007.02.023
Reference: VETMIC 3601
To appear in: VETMIC
Received date: 16-11-2006 Revised date: 7-2-2007 Accepted date: 19-2-2007
Please cite this article as: Vretou, E., Radouani, F., Psarrou, E., Kritikos, I., Xylouri, E., Mangana, O., Evaluation of two commercial assays for the detection of Chlamydophila abortus antibodies, Veterinary Microbiology (2007), doi:10.1016/j.vetmic.2007.02.023 This is a PDF file of an unedited manuscript that has been accepted for publication.
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Accepted Manuscript
Evaluation of two commercial assays for the detection of
1
Chlamydophila abortus antibodies
2 3 4 5
Running title: Serological diagnosis of ovine enzootic abortion 6
7 8
E. Vretou
*, F. Radouani, E. Psarrou, I. Kritikos, E. Xylouri
1, O. Mangana
29
10
Department of Microbiology, Hellenic Pasteur Institute, 127 Avenue Vassilissis 11
Sofias, Athens 115 21, Greece 12
1
Agricultural University of Athens, Iera Odos 75, 118 55 Athens 13
2
Veterinary Institute for Infectious and Parasitic Diseases, 153 10 Athens 14
15 16 17 18
*Corresponding author. Telephone: (30) 210 64 78 873. Fax: (30) 210 98 11 884 E- 19
mail: vretou@mail.pasteur.gr 20
21
22
Accepted Manuscript
ABSTRACT 1
Two commercial enzyme-linked immunosorbent assays (ELISA), the 2
CHEKIT®-CHLAMYDIA which uses inactivated C. psittaci antigen, and the 3
Chlamydophila abortus ELISA produced by the Institut Pourquier which uses a 4
recombinant fragment of the 80-90 kDa protein, were evaluated with the objective to 5
determine whether the new ELISAs would perform as improved alternatives to the 6
complement fixation test (CFT) for the serological diagnosis of ovine enzootic 7
abortion (OEA). The results were compared to those obtained by the CFT and the 8
competitive ELISA (cELISA). The tests were assessed with a panel of 17 serum 9
samples from specific pathogen-free lambs experimentally infected with various 10
subtypes of C. pecorum, with sera from 45 C. abortus-infected pregnant sheep and 11
from 54 sheep free of OEA. The Chlamydophila abortus ELISA was identified as 12
being more specific and sensitive than the other tests. The 4 assays were evaluated 13
further with 254 sera from flocks with documented OEA, from flocks with no history 14
of abortion, and from animals after abortion of unknown cause. The Chlamydophila 15
abortus ELISA by the Institut Pourquier identified less OEA-positive sera than the 16
other assays though it identified correctly 9 of 10 OEA-positive flocks. The basis of 17
the discordant results is discussed.
18 19 20 21
Key words: ovine enzootic abortion; Chlamydophila abortus; Chlamydophila 22
pecorum; serological diagnosis; indirect ELISA 23
24
25
Accepted Manuscript
1 2
INTRODUCTION 3
Chlamydophila abortus (C. abortus) is a zoonotic pathogen that induces 4
infectious abortion in ruminants, termed Ovine Enzootic Abortion (OEA) and is also 5
hazardous for pregnant women ( Longbottom and Coulter, 2003). OEA is the most 6
common disease of reproductive failure in sheep- and goat-breeding countries in 7
Europe and has an important economic impact (Aitken, 1993). Infected pregnant ewes 8
and goats abort late in gestation or give birth to weak lambs as a result of affected 9
placenta. Bacteria excreted at abortion are the main source of infection of susceptible 10
animals through ingestion or inhalation of the uterine discharge. After the onset of 11
abortion, most of the animals acquire immunity and rebreed successfully. Chronically 12
infected animals that excreted detectable amounts of chlamydial antigen during 13
subsequent estrous cycles and maintained a persistent specific antibody response for 14
up to a period of 2.5 years were observed after experimentally induced chlamydial 15
abortion. (Papp et al., 1994).
16
Early diagnosis of chlamydia as the cause of abortion is important to prevent 17
and limit the spread of infection. Though direct evidence of the pathogen is the 18
ultimate diagnosis, serological assays are more suitable for screening large numbers.
19
The complement fixation test (CFT) is the most widely used test and is recommended 20
by the Organisation Internationale des Epizooties (OIE) (www.oie.int). However, it 21
lacks specificity due to its antigen, consisting mainly of the heat-resistant 22
lipopolysaccharide (LPS), common to all Chlamydiaceae species (Brade et al., 1987).
23
Ruminants in particular are also largely infected by Chlamydophila pecorum (C.
24
pecorum), subspecies of which cause a variety of clinical manifestations like arthritis,
25
Accepted Manuscript
conjunctivitis and enteric infections (Kaltenboeck et al., 1993; Fukushi and Hirai, 1
1992; Anderson et al., 1996).
2
Several experimental assays have been developed in order to improve 3
chlamydial serology. These include indirect immunofluorescence (Markey et al., 4
1993), immunoblotting (Jones et al., 1997), and indirect enzyme-linked immunoasays 5
(ELISAs) using whole elementary bodies (EB) or extracts thereof (Cevenini et al., 6
1989; Markey et al., 1993; Anderson et al., 1995) and purified or recombinant LPS 7
(Sting and Hafez, 1992; Griffiths et al., 1996). More specific assays are based on the 8
major outer membrane protein (MOMP, Kaltenboeck et al., 1997; Hoelzle et al., 2004;
9
Salti-Montesanto et al., 1997; Gut-Zangger et al., 1999; Borel et al., 2004), and the 10
polymorphic outer membrane proteins (POMP) at 80-90kDa (Buendia et al., 2001, 11
Longbottom et al., 2001 and 2002). Few of these assays have gone beyond 12
experimental “in house” application, causing urgent need for harmonization. In the 13
present study we assessed the performance of two commercial ELISAs, the 14
CHEKIT®-CHLAMYDIA which uses inactivated C. psittaci antigen, and the “ELISA 15
Chlamydophila abortus” produced by the Institut Pourquier which uses a recombinant 16
fragment of an 80-90 kDa protein. The results were compared to those obtained by the 17
CFT as the primary reference assay and the “in house” competitive cELISA (Salti- 18
Montesanto et al., 1997).t. The objective was to determine whether the new ELISAs 19
would perform as improved alternatives to CFT for the serological diagnosis of OEA.
20 21 22 23
MATERIALS AND METHODS 24
Antibody assays.
25
Accepted Manuscript
(i) Chlamydophila abortus ELISA (version P00700/04-18/02/05; Institut Pourquier, 1
Montpellier, France) was performed according to the instructions of the manufacturer.
2
The final values were expressed as Sample/ Positive control % (S/P %). Sera with 3
S/P% equal to or lower than 50% were considered negative, sera with an S/P%
4
between 50-and 60% were doubtful, and sera with an S/P% > 60% were positive for 5
OEA-infection.
6
(ii) CHEKIT®-CHLAMYDIA ELISA (Dr. Bommeli AG-IDEXX, Switzerland) results 7
were normalized using the positive and negative control sera and were expressed as 8
Value (%). Sera with a value below 30% were considered negative, sera with values 9
between 30-40% were ambiguous, while sera > 40% were considered positive for 10
OEA-infection according to the instructions in the kit.
11
(iii) Competitive enzyme-linked immunosorbent assay (cELISA).The assay was 12
performed essentially as described previously and the results were expressed as % 13
inhibition (Salti-Montesanto et al., 1997). Sera with inhibition values lower than 50%
14
were considered negative for OEA. The questionable zone which was originally 15
included in the calculations was omitted from this study for purposes of conformity 16
with the other ELISAs. The selected cut-off predictably increased the specificity and 17
reduced the sensitivity of the test.
18
(iv) Complement Fixation Test (CFT). The CFT was performed according to 19
published procedures (Stamp et al., 1952) using a Greek C. abortus strain as antigen, a 20
twofold serum dilution, and standard reagents (Virion/Serion, Würzburg, Germany).
21
CF titers were expressed as the highest dilution of serum with less than 50% lysis of 22
red blood cells. A CFT titer equal or greater than 1:40 was positive.
23
All absorbance measurements were performed at 450 nm using a microtiter plate 24
reader (Dynatech, Quernsey, UK). Doubtful (Pourquier), ambiguous (CHEKIT), or
25
Accepted Manuscript
equivocal (CFT) results were considered positive for the calculation of concordance 1
between the tests.
2 3
Serum samples 4
Experimental sera 5
The experimental sera (groups 1, 2 and 3) were produced in the Moredun Research 6
Institute, Scotland, UK, according to published protocols (Jones et al., 1995 and 1997) 7
and were provided to us for the evaluation of serological OAE-tests. Group 1 consisted 8
of 17 samples of 11 specific pathogen-free (SPF) lambs immunized with various C.
9
pecorum isolates: 2 with the conjunctival isolate 84-796 (4 sera), 3 with the arthritic 10
isolate P787 (6 samples) and 4 with the enteric isolate 84-604 or W73 (5 samples).
11
Two more samples were from 2 SPF sheep immunized with the Chlamydia suis strain 12
S45. Group 2 comprised sera from 45 sheep in which OAE had been experimentally 13
reproduced by infection with strain S26/3. Within this group 33 sera were from ewes 14
that developed heavily infected placentas or aborted (group 2A) and 12 samples were 15
from ewes that lambed and developed lightly infected or culture-positive placentas 16
(group 2B). Group 3 comprised sera from 54 Scottish sheep which were known 17
through their participation in the Premium Health Scheme for Sheep to be clinically 18
free of OEA and which were used as negative controls in experimental trials.
19
Field sera 20
Group 4 comprised 97 serum samples from 10 flocks of Greek sheep and goats with 21
known clinical history of OEA. OEA was documented by positive Gimenez stain on 22
aborted placentas and isolation of chlamydia. Group 5 comprised 69 samples from 8 23
mixed flocks with no previous clinical history of chlamydial abortion. Group 6 24
consisted of 26 serum samples from an OEA free flock from Scotland in which
25
Accepted Manuscript
infection caused by a Cp. pecorum arthritis/conjunctivitis subtype was unconfirmed 1
but suspected. Group 7 consisted of 62 serum samples from ewes and goats, 2
originating from 12 Greek organic farms. Sampling was during spring of 2005 from 3
flocks with incident of abortions during the reproductive period of 2004-2005. All 4
sampled animals had aborted in the last trimester of pregnancy or had given stillbirth.
5
Flocks were defined as positive if at least one individual serum sample in the flock was 6
positive according to the criteria of the respective test.
7
Statistical analysis. Standard procedures were used to calculate the sensitivity, 8
specificity (free software WinEpiscope 2.0 http://www.clive.ed.ac.uk).
9 10
RESULTS 11
Experimental sera 12
The analytical specificity of the two commercial indirect ELISAs, “ELISA 13
Chlamydophila abortus” from the Pourquier Institute (named here Pourquier ELISA) 14
and “ CHEKIT®-CHLAMYDIA ” (named here CHEKIT-ELISA) and of the cELISA and 15
the CFT was assessed with a panel of 15 sera derived from SPF animals immunized 16
with different subspecies of C. abortus and 2 from SPF lambs infected with Chlamydia 17
suis (group 1, Table 1 and Figure 1). The Pourquier ELISA was highly specific since 18
all 17 sera were negative apart from a borderline reactivity (S/P value 50.1 %) with a 19
serum from a lamb infected with a conjunctivitis- subtype of C. pecorum. The 20
CHEKIT ELISA reacted with 5 C. pecorum sera of the conjunctivitis and the 21
arthritogenic subspecies but with none of the enteric subtype. Furthermore, this ELISA 22
reacted with one serum from a lamb immunized with Chlamydia suis which was also 23
positive by the CFT. In total, 8 out of the 17 sera in group 1 were positive by CFT.
24
None of the sera had an inhibition value above 50% by the cELISA confirming
25
Accepted Manuscript
previous results (Salti-Montesanto et al., 1997), and none of the 4 tests reacted 1
positively with the serum of an unvaccinated SPF sheep (not shown).
2
Performance characteristics of the indirect ELISAs The sensitivity and specificity 3
of the 2 commercial tests was determined with a panel of 45 serum samples from 4
sheep experimentally infected with C. abortus (group 2A-B) and 54 sera from OEA- 5
free sheep (group 3). In terms of specificity the Pourquier ELISA proved as the most 6
specific assay (100% specificity) since it did not react with any of the OEA-free sera in 7
group 3 (Table 2 and Figure 1). The CHEKIT-ELISA identified 52 of the 54 negative 8
sera (specificity 96.3%). One serum had an inhibition value above 50 % in the cELISA 9
(specificity 98.1%), while a specificity of 88.9% was calculated for the CFT which 10
reacted positively with 6 sera.
11
In group 2A, which comprised sera from ewes with abortion and heavily infected 12
placentas, 23 out of the 33 sera (69.7%) were positive by the 4 tests. One serum was 13
found negative by all 4 assays though the animal had aborted and placental 14
examination showed severe lesions. In group 2B in which ewes had lambed but their 15
placentas were positive by cell culture, the 4 tests agreed in 3 positive out of 12 sera 16
only (25%). As presented in Table 2 the Pourquier ELISA and the cELISA performed 17
similarly in the 2 groups yielding 78.8 % and 81.8% positives in group 2A, and 83.3%
18
and 66.7% in group 2B, respectively. In contrast, the LPS-based tests detected more 19
positive sera in the animal group with abortions (87.9%) compared to the scores in 20
group 2B, which were 33.3% and 16.6% for the CHEKIT-ELISA and the CFT, 21
respectively. Based on the results with groups 2A+2B the sensitivities of the tests 22
were 80% for the Pourquier-, 73.3% for the CHEKIT-ELISA, 68.8% or the CFT and 23
77.7% for the cELISA.
24
Field sera
25
Accepted Manuscript
The performance of the 2 new ELISAs, Pourquier and CHEKIT was further 1
evaluated with a panel of 254 field sera, groups 4-7 (Table 3, 4 and Figure 1). Group 4 2
consisted of 97 sera from 10 Greek flocks with documented history of OEA. The CFT 3
identified 49 positives (50.5%) and 18 sera as equivocal .The CHEKIT ELISA 4
detected 28 positives (28.8%) and 2 more sera as ambiguous, while 29 sera (29, 8%) 5
were positive by the cELISA exceeding 50% inhibition. These results were in contrast 6
to the results of the Pourquier ELISA which detected only 14 positive samples (14.4 7
%) and 3 more as doubtful. These 14 positive scores, however, identified correctly 9 8
out of the 10 flocks as positive (1-2 positives per flock) and missed one flock (#3, 9
Table 4). Forty one sera were negative in all 4 tests and 42 sera were negative in the 10
Pourquier-ELISA but positive in other assays. The analysis of these 42 sera with 11
discordant results as well as and their distribution among the flocks is presented in 12
Table 4. The results showed that the test had a lower sensitivity when examining field 13
sera, since all 42 Pourquier-ELISA-negative sera were positive in one, two or three 14
other assays combined. The CHEKIT-ELISA identified 7 of the 10 flocks. Flock #9 15
was clearly negative; flocks #3 and 5 had a single ambivalent score. All 10 OEA- 16
positive flocks tested positive by the CFT and the cELISA.
17
Group 5 comprised 69 field samples from 8 mixed flocks with no previous 18
history of chlamydial abortion. Fifteen samples were CFT-positive (21.7%) and 21 19
further samples gave equivocal results. Six sera were positive (8.6%) by the CHEKIT- 20
ELISA and 4 more tested ambiguous. Overall, four flocks were positive by either the 21
CFT or the CHEKIT-ELISA, and two of them were in concordance. None of the sera 22
in this group tested positive by the Pourquier-ELISA and the cELISA.
23
Group 6 consisted of 26 serum samples from an OEA-free flock from Scotland 24
in which infection caused by C. pecorum of the arthritis/conjunctivitis subtype was
25
Accepted Manuscript
unconfirmed, but suspected. Six sera (23%) were positive by the CFT and 4 were 1
equivalent (Table 3). The CHEKIT ELISA identified 3 false positives (11.5%), 2
contrary to the Pourquier and the cELISA which did not react with any of the sera in 3
this group.
4
Group 7 consisted of 62 ewes and female goats originating from 12 Greek 5
organic farms which had aborted during the reproductive period 2004-2005. The 6
Pourquier ELISA and the CFT identified 8 positives (12.9%) and the CHEKIT ELISA 7
15 positives (24.1 %). Surprisingly, more than twice this number tested positive by the 8
cELISA (35 sera, 56.5%). Agreement between the 4 assays was observed in 6 flocks, 3 9
positive and 3 negative. One further flock was negative by the Pourquier-ELISA and 10
positive by the 3 tests and a further one that was negative by the 3 tests was positive by 11
the CHEKIT-ELISA. Ten of the 44 sera among the other flocks were negative by the 4 12
assays while 28 sera had discordant results between the Pourquier-ELISA and the 13
other tests.
14
The original results of the 3 ELISAs: Pourquier, CHEKIT-and cELISA and 15
their respective cut-offs at values 60% and 40% for the Pourquier- and the CHEKIT 16
ELISA, and 50% inhibition for the c-ELISA, are shown in Figure 1. The values by the 17
Pourquier-ELISA for the OEA-free groups 3, 5 and 6 were well clustered but far 18
below the cut-off line at 60% suggested by the manufacturer. We therefore calculated 19
the cut-off values for the 3 tests based on the mean value of the OEA-free reference 20
sera (group 3) + 3X Standard Deviation. The resulting value was 22.4% for the 21
Pourquier-ELISA, considerably lower than the suggested cut-off, contrary to the 22
CHEKIT-ELISA which was 51.8 %, exceeding the suggested cut-off of 40%. The 23
calculated cut-off for the cELISA of 46.1% was close to the 50% inhibition value set 24
as the threshold of the test in this study.
25
Accepted Manuscript
The concordances between the four tests are presented in Table 5. In general, the 1
concordances between tests were higher in the experimental than in the field sera. The 2
overall agreement between Pourquier- and cELISA at 84.0% was moderate (kappa = 3
0.54) as it was between the Pourquier- and the CHEKIT-ELISA (82.9%, kappa= 0.52).
4
The kappa-values between Pourquier-ELISA and CFT, CHEKIT and CFT, cELISA 5
and CFT and CHEKIT and cELISA were 0.25, 0.36, and 0.31 and 0.42, respectively, 6
suggesting significant differences between the assays.
7 8
DISCUSSION 9
Recent development of tests for the improvement of the serological diagnosis 10
of OEA has focused mainly on three surface exposed, immunodominant antigens, the 11
LPS, the MOMP and the POMPs. Given that all Chlamydiaceae species share the 3- 12
deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) trisaccharide (Brade et al., 1987), 13
the use of LPS in natural or recombinant form was not considered as the most 14
appropriate antigen for the serodiagnosis of OEA (Griffiths et al., 1996). Interestingly, 15
C. psittaci contains in addition to the Kdo -trisaccharide a branched tetrasaccharide 16
(Brabetz et al., 2000) as reported by Brade’s laboratory, which has also produced an 17
anti-glycolipid mAb capable of differentiating between C. psittaci and C. pecorum 18
(Mueller-Loennies et al., 2006). Alternatively, the MOMP VS1 and 2 sequences 19
revealed a great deal of diversity between C. abortus and C. pecorum strains providing 20
the molecular basis for the development of a specific competitive ELISA (Salti- 21
Montesanto et al., 2007). The POMPS finally, have been shown to be important 22
serodiagnosis tools with improved specificity (Buendia et al., 2001; Longbottom et al., 23
2001 and 2002; Livingstone et al., 2005).
24
Accepted Manuscript
In this study we have evaluated the performance of two commercial ELISAs, 1
the CHEKIT®-CHLAMYDIA which uses inactivated C. psittaci antigen, and the 2
Chlamydophila abortus ELISA produced by the Institut Pourquier which uses a 3
recombinant fragment of the 80-90 kDa (POMP) protein, with the objective to 4
determine whether the new ELISAs would perform as improved alternatives to CFT 5
for the serological diagnosis of OEA. The results indicate that the POMP-based 6
Pourquier-ELISA could do so. The test was highly specific since it did not react with 7
any of the sera from the SPF-lambs experimentally infected with various subtypes of 8
C. pecorum in contrast to the CHEKIT-ELISA (Table 1). Furthermore, it did not 9
produce any false positive results with reference sera known to be free from OEA 10
(Table 2), or with sera from a flock with a suspected C. pecorum infection, or with sera 11
from flocks with no clinical history of abortions (Table 3).
12
When tested with sera from pregnant ewes after experimental infection with C.
13
abortus the Pourquier-ELISA proved to be more sensitive than the CHEKIT-ELISA, 14
the CFT and the cELISA. Moreover, while it identified fewer positive sera from ewes 15
after abortion (group 2A), the test detected more positive sera from infected ewes 16
lambing normally than any other assay (group 2B, Table 2). These results were in 17
agreement with recent data reporting that the anti-rOMP90-4 response was lower in the 18
animals that aborted than that observed in the animals that lambed normally 19
(Livingstone et al., 2005). When tested with field sera from flocks with documented 20
OEA, however, the performance of the Pourquier-ELISA was less satisfactory, though 21
it identified 9 out of 10 OEA-positive flocks. Only one ewe had aborted in this 22
particular flock, suggesting that the spread of the disease was limited. At the individual 23
serum level, however, only half of the sera detected by the CHEKIT or the cELISA 24
were positive by the Pourquier-ELISA. Furthermore, 42 of the 83 Pourquier-ELISA-
25
Accepted Manuscript
negative sera were positive in other assays. Similar results were also obtained with sera 1
from aborted goats (group 7). A possible explanation for the lower sensitivity of the 2
Pourquier –ELISA may lie in the choice of the particular recombinant POMP fragment 3
used in the assay. Fragmentation of the whole POMP90 molecule in overlapping 4
recombinant antigens and their assessment with a similar panel of sera had revealed 5
considerable variation in the sensitivity among the different fragments. Furthermore, 6
fragment rOMP90-3 which was as sensitive as rOMP90-4 when tested with 7
experimental sera was half as sensitive as rOMP90-4 when tested with field sera 8
((Longbottom et al., 2002). A further possible explanation is that the selected cut-off 9
value at 60% S/P, which provides the assay with a good specificity, appears relatively 10
high considering the values in the negative groups 3, 5 and 6, that did not exceed 30%
11
(Figure 1). Alternatively, one could assume that the particular POMP-fragment used as 12
antigen might vary in the abortus-subtypes present in the field, and that these variants 13
would raise less-reactive sera in the infected animals.
14
There is an urgent requirement for standardized serological tests for the indirect 15
diagnosis of OEA in order to ensure comparability and harmonization among 16
European veterinary laboratories. In addition, there is a need for new tests capable to 17
differentiate between naturally infected and vaccinated animals. None of the tests 18
under study has this potential. The requirements for any new test are greater sensitivity 19
and specificity than that of the CFT. For these criteria, the CHEKIT-ELISA offers the 20
advantage of easy and speedy performance but it is based on a less well defined 21
antigen containing lipopolysaccharide. The data presented here show that the 22
Pourquier-ELISA is a valuable test for the specific serological diagnosis of OEA. They 23
further suggest that modifying the cut-off to increase sensitivity would be more 24
favorable.
25
Accepted Manuscript
Acknowledgements 1
The commercial diagnostic kits were kindly provided by the manufacturers. Dr F.
2
Radouani, Institut Pasteur du Maroc, received a fellowship from the “Reseau des 3
Instituts Pasteur”.
4
5
Accepted Manuscript
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Table and Figure Legends 1
2
Table 1 Analytical specificity of the 4 assays with sera from SPF lambs immunized 3
with various strains of C. pecorum and with Chlamydia suis.
4 5
Table 2 Reactivity of the 4 tests with experimental positive and negative reference 6
sera 7
8
Table 3 Performance of the 4 tests with field sera (groups 4-7) 9
10 11
Table 4 Analysis of the discordant results in 10 flocks with documented OEA 12
(group 4) 13
14 15
Table 5 Concordance between the results obtained with the four assays for 16
experimental and field sera 17
18
Figure 1 19
Original results of the 3 ELISAs: Pourquier- (top), CHEKIT-ELISA (middle) and 20
cELISA (bottom). Sera 1-17 from SPF lambs immunized with various pecorum 21
strains group1; sera 18-50 and 51-62 from experimentally infected ewes, group 2A 22
and 2B; sera 63-116 from documented OEA-free sheep, group 3; sera 117-213 from 23
OEA-infected flocks, group 4; sera 214-282 from OEA-free flocks, group 5 ; sera 24
283-308 from a flock infected with C. pecorum, group 6:, sera 309-370 from ewes 25
after abortion group 7. The respective cut-offs of the 3 tests are shown as dashed 26
horizontal lines.
27
Accepted Manuscript
-10,0 10,0 30,0 50,0 70,0 90,0 110,0 130,0 150,0
-20 30 80 130 180 230 280 330 380
value (%)
-20,0 10,0 40,0 70,0 100,0 130,0 160,0 190,0 220,0
-20 30 80 130 180 230 280 330 380
value (%)
-10,0 10,0 30,0 50,0 70,0 90,0 110,0
-20 30 80 130 180 230 280 330 380
serum number
inhibition (%)
Figure 1
Figure 1
Accepted Manuscript
Table 1 Analytical specificity of the 4 assays with sera from SPF lambs immunized with various strains of C. pecorum and with Chlamydia suis.
Test Number of positives
conjunctival (4)
aarthritogenic
(6) enteric
(5) porcine
(2)
Pourquier 0
b(1)
c0 0 0
CHEKIT 2 2 (1) 0 1
CFT 4 3 0 1
cELISA 0 0 0 0
a
Number of sera tested;
bnumber of positive
cnumber of doubtful (Pourquier) or ambiguous (CHEKIT) results
Table 1
Accepted Manuscript
Table 2 Reactivity of the 4 tests with experimental positive and negative reference sera
Groups 2&3 Pourquier CHEKIT CFT cELISA
pos (%) pos (%) pos (%) pos (%)
2A (N=33) 26 78.8 29
a(1)
b87.9 29(2) 87.9 27 81.8
2B (N =12) 10 83.3 4(1) 33.3 2(3) 16.6 8 66.7
Group 2A+B (N=45) 36 80 33(2) 73.3 31(5) 68.8 35 77.7
Group 3 (N= 54) 0 100 1(1) 96.3 2(4) 88.9 1 98.1
a
number of positive;
bnumber of ambiguous (CHEKIT) and equivocal results (CFT).
Table 2
Accepted Manuscript
Table 3 Performance of the 4 tests with field sera (groups 4-7)
a
number of positive
bnumber of doubtful (Pourquier), ambiguous (CHEKIT) or equivocal results (CFT).
group 4- naturally OEA-infected flocks; group 5- flocks with no history of abortion; 6- one flock with suspected infection by a C. pecorum arthritis/conjunctivitis-subtype ; 7- ewes/goats from organic farms which had late abortions or had given stillbirth of unknown cause
Group 4 N= 97, 10 flocks
Group 5 N= 69, 8 flocks
Group 6 N= 26, 1 flock
Group 7 N= 62, 12 flocks
Test No of
positive sera (%) No of positive flocks
No of
positive sera (%) No of positive flocks
No of positive
sera
(%) No of
positive sera
(%) No of
positive flocks
Pourquier 14
a(3)
b14.4 9 0 0 0 0 0 8(1) 12.9 3
CHEKIT 28(2) 28.8 7 6(4) 8.6 4 3 11.5 15(1) 24.1 7
CFT 49(18) 50.5 10 15(21) 21.7 4 6(4) 23 8 12.9 6
cELISA 29 29.8 10 0 0 0 0 0 35 56.5 8
Table 3
Accepted Manuscript
Table 4 Analysis of the discordant results in 10 flocks with documented OEA (group 4) Flock # N
bDiscordant
Results
c+ve in
3 tests +ve in
CHEKIT +CFT +ve in
CFT +cELISA +ve
in CHEKIT +ve
in CFT +ve
in cELISA
1
a19 4 1 2 1
2 6 1 1
3
a7 4 3 1
4
a7 4 1 1 1 1
5 10 7 5 2
6
a15 9 3 1 3 2
7
a6 4 3 1
8 10 4 1 2 1
9 6 1 1
10 11 4 1 1 2
a
