Comparison of three commercial serological tests for the detection of Chlamydia abortus infection in ewes

Comparison of three commercial serological tests for the detection of Chlamydia abortus infection... Background: 20.9% of diagnosable abortions in Ireland in 2015 were caused by Chlamydia abortus infection. Abortion usually occurs in the last 2–3 weeks of gestation, and up to 30% of ewes may be affected in naïve flocks. Serological diagnosis of EAE in flocks using LPS or whole bacteria as antigens is often hindered by cross reactions with C. pecorum. Although the complement fixation test is the official test for diagnosis of EAE, more sensitive and specific ELISA based tests have been developed. This study aimed to compare three commercial ELISA kits to detect C. abortus antibodies in ewes and to determine which of the kits had the highest sensitivity. The IDvet kit utilises a MOMP peptide antigen, the MVD-Enfer kit is based on a POMP90–3 antigen while the LSI kit plates are coated with chlamydial LPS. The study also aimed to examine the potential of these ELISAs to distinguish infected animals that go on to abort compared to those that have live lambs. Ewes were vaccinated with either a commercial live vaccine (n = 10) or Tris-buffer sham inoculation (n = 9) 5 months prior to gestation, these ewes were then challenged with C. abortus (1 × 10 IFU/ml) on day 90 of gestation. Sera were collected at pre- vaccination, 14 days post vaccination, 35 days post vaccination, pre-challenge, 35 days post challenge and 3 weeks post lambing/abortion (~ 70 days post challenge) and tested using the 3 aforementioned ELISAs to determine if one ELISA was more sensitive at detecting circulating anti-chlamydial antibodies. Results: Sensitivity was highest with the LSI test kit at 94.74%, followed by the MVD-Enfer and IDvet kits, at 78.95 and 73.68% respectively. Ewes vaccinated with Enzovax became seropositive at 14 days post vaccination with all kits. Following challenge at day 90 of gestation, antibody titres steadily rose in all groups of ewes. With all ELISA kits, antibody levels were higher in ewes that aborted compared to ewes that had live lambs at 35 days post challenge and three weeks post lambing, and statistically significantly higher antibody levels were recorded in ewes that aborted compared to ewes that had live lambs using the MVD-ENFER ELISA at three weeks post lambing (P = 0.0482). Conclusions: The LSI assay was the most sensitive out of the three kits tested in this study, when sera were tested at three weeks post lambing. As the LPS used in this kit is cross-reactive with all chlamydia, it is good for identifying flocks infected with any chlamydial species, but it is not considered specific for C. abortus. Furthermore, antibody levels were higher in ewes that aborted compared to ewes that had live lambs, at both 35 days post challenge and at three weeks post lambing. Future work should include evaluation of a larger number of sera at a wider range of time-points as well as an estimation of the specificity of commercially available assays. Keywords: Chlamydia abortus, Enzootic abortion of ewes, ELISA sensitivity, ELISA comparison * Correspondence: lauren.oneill@ucd.ie L. M. O’Neill and Á. O’Driscoll contributed equally to this work. School of Veterinary Medicine, University College Dublin, Room 07A, Belfield, Dublin 4, Ireland © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 2 of 9 Background potential of the different kits to identify ewes that subse- Enzootic abortion of ewes (EAE) is the second most quently progress to abortion. common infectious cause of ovine abortion in Ireland, causing 20.9% of diagnosable abortions in 2015 [1]. The Method causative agent, Chlamydia abortus, is a serious zoonotic Sera from two groups of ewes (n = 19) were tested using pathogen in pregnant women which makes accurate three different commercial ELISA kits: IDvet, LSI and testing essential in the diagnosis and control of infected MVD-Enfer. The sera were generated as part of a sheep [2]. The disease manifests itself as a placentitis, vaccination trial [13]. This trial involved 5 different leading to abortion in the last 2 to 3 weeks of gestation treatment groups including the testing of novel recom- [3]. Aborting ewes are the principal means of transmis- binant protein vaccines against EAE. The sera from the sion of infection but rarely show prior clinical signs. As positive control ewes that received a live commercial a result early, detection of infection during pregnancy is vaccine and the negative control ewes that received a vital in permitting therapeutic intervention and control- sham Tris-buffer inoculation were used in the current ling the spread of disease. study. In summary, ten ewes were vaccinated 5 months Serological diagnosis of EAE in flocks is challenging prior to mating with Enzovax (MSD Animal Health), the for a number of reasons. When serological tests such as commercial vaccine used in Ireland, while the nine were the complement fixation test (CFT) use LPS or whole given a sham inoculation of Tris-buffer (control ewes). bacteria as antigens in the diagnosis of C. abortus infec- All the ewes were challenged by subcutaneous inoculation tion, specificity tends to be low as cross reactions with at day 90 of gestation with 10 IFU of C. abortus isolate Chlamydia pecorum are often observed [4]. Chlamydia C95/27. All sera were tested at six time-points: 1 = pre- pecorum mainly causes inapparent enteric infections [5], vaccination, 2 = 14 days post vaccination, 3 = 35 days post but is also known to cause arthritis, conjunctivitis and vaccination, 4 = pre-challenge, 5 = 35 days post challenge pneumonia [3]. Cross-reactive antibodies may arise from and 6 = three weeks post lambing. All sera were thawed at other chlamydial species and certain Gram-negative room temperature while the kit reagents were brought to bacteria such as Acinetobacter spp. [4]. Thus, specific room temperature. The ELISAs were carried out accord- diagnostic tests are required to properly understand the ing to manufacturers’ instructions. The format of each epidemiology of EAE and in implementing control ELISA tested was an indirect ELISA. The S/P% was calcu- strategies. The diagnosis of EAE is further complicated lated for each sample to determine the titres of antibodies by the ability of C. abortus to cause latent infections [6]. using the formula [OD -OD ]/ [OD sample negative control positive Pathological lesions in the ovine placenta do not usually -OD ] × 100. For the LSI kit, a titre >25 control negative control develop until day 90 of gestation [7]. S/P% was considered positive. For the IDvet kit, an S/P% The CFT is the most widely used technique for ≤50% was considered negative, an S/P% greater than 50% serological diagnosis of EAE and is considered the gold and less than 60% was considered doubtful and an S/ standard test for official trade purposes [8]. However, P%≥60% was considered positive. For the MVD-Enfer kit, advances have been made in the development of more an S/P% ≤20% was considered negative, an S/P% greater sensitive and specific tests such as ELISAs that specific- than 20% and less than 30% was considered doubtful and ally detect antibodies to a range of chlamydial antigens an S/P% ≥30%was considered positive. including major outer membrane protein (MOMP) and polymorphic outer membrane proteins (POMP) [9]. Statistical analysis The aim of this study was to compare three commer- Statistical analysis of results was conducted on Graph- cial ELISAs designed to detect antibodies to C. abortus Pad Prism 7.03. Parametric unpaired t tests were carried in ewes. Each ELISA utilises a different chlamydial anti- out. Sensitivities (Se) of the kits were calculated using gen. In the IDvet kit, wells are coated with a MOMP the results from three weeks post lambing and the fol- peptide antigen, which is known to have genus-, species- lowing formula: , subspecies- and serotype-specific immunodomains The number of diseased animals testing positive [10]. Wells of the MVD-Enfer kit are coated with a Se ¼ The total number of diseased animals POMP90–3 antigen considered to be specific for C. abortus [11]. The LSI kit uses chlamydial LPS as the The agreement between the ELISAs was determined antigen coating the wells. This is a genus-specific anti- from the Kappa coefficient (κ) and percentage concord- gen common to all chlamydial species [12]. ance for samples from all time points [14] using Epi- The study aimed to determine which of the three kits Tools epidemiological calculators [15]. Concordance was had the highest sensitivity and to evaluate the influence calculated as the sum of positive –positive values and of the different antigens used in the assays on test char- negative-negative values expressed as a percentage of the acteristics. Furthermore, the study aimed to evaluate the total number of serum samples [16]. O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 3 of 9 Results following challenge. Ewes 3376, 3554 and 4901 had an All ELISA kits detected a similar trend in antibody levels S/P% of 1.18, 1.34 and 6.22 respectively, at 3 weeks post post-vaccination and post-challenge. The antibody levels lambing. For the other ewes there was an increase in in the two groups of ewes at the six time-points are dis- antibody levels after vaccination and again after chal- played in Figs. 1, 2, 3, 4, 5 and 6. The ewes vaccinated lenge at day 90 of gestation. with the commercial vaccine showed a rise in antibody The results of the MVD-Enfer kit for each individual titre after vaccination (Figs. 1, 3 and 5). This is to be Tris-buffer ewe are shown in Fig. 2. As expected, all expected as it is a live vaccine and the animals were ewes tested negative following the sham inoculation. At therefore exposed to the full range of chlamydial anti- 35 days post challenge some ewes begin to test positive, gens. Antibody levels in both groups rose after challenge with all ewes in the group testing positive at 3 weeks at day 90 of gestation (Figs. 1, 2, 3, 4, 5 and 6). post-lambing. Ewe 3547 tested doubtful at 35 days post All ewes showed a steady rise in antibody levels after challenge with an S/P% of 24.48, but this ewe subse- challenge, however the Enzovax vaccinated ewes at day quently tested positive at 3 weeks post lambing. 35 post challenge appeared to plateau (Figs. 1 and 3) The antibody levels in individual ewes from the Enzo- unlike Tris-buffer vaccinated ewes (Figs. 2 and 4). It is vax group using the IDvet kit are shown in Fig. 3.In important to note that in general the IDvet kit recorded general, antibody levels rose following vaccination. How- higher antibody levels for both vaccine groups and ever, four ewes did not test positive at either 14 or peaked at levels higher than 150 S/P% (Fig. 3). The other 35 days post vaccination. Ewe 3376 tested doubtful at ELISA kits did not exceed 150 S/P% (Figs. 1 and 5). day 14 with an S/P% of 51.17 and tested negative at day All Enzovax ewes after vaccination demonstrated a 35 with an S/P% of 25.19. Ewe 3554 tested negative at peak similar to that post-challenge, showing the diffi- day 14 and day 35, with an S/P% of 23.33 and 19.18 re- culty of differentiating vaccinated ewes from infected spectively. Ewe 4773 also tested negative at day 14 and ewes using the current commercial vaccine and available day 35, with an S/P% of 49.81 and 25.21 respectively. ELISAs. Ewe 4901 tested doubtful at day 14 with an S/P% of 56. The results of the MVD-Enfer kit for each individual 90 and tested negative at day 35 with an S/P% of 17.31. Enzovax vaccinated ewe are detailed in Fig. 1. At day 14 Following challenge, there was a general rise in antibody post vaccination, four ewes tested negative and one ewe levels, although four ewes did not fit this trend. At tested doubtful. Ewe number 3376 tested doubtful with 3 weeks post lambing, ewe 3543 tested doubtful, with an an S/P% of 26.69. Ewes 3543, 3554, 4773, 4901 all tested S/P% of 53.01. Furthermore, ewes 3554, 4749 and 4773 negative, with an S/P% of 6.39, 2.56, 12.23 and 15.66 re- all tested negative at 3 weeks post lambing, with an spectively. These five ewes all tested negative at day 35 S/P% of 9.04, 25.49 and 8.29 respectively. post vaccination. Three ewes tested negative at both The results of the IDvet kit for Tris-buffer ewes are 35 days post challenge and 3 weeks post lambing, so shown in Fig. 4. All ewes tested negative in the vaccine antibody levels of these ewes did not show an increase phase of the study. Most ewes in the group tested Fig. 1 Enzovax ewes tested with MVD-Enfer ki. MVD-Enfer kit showing antibody levels in ewes vaccinated with Enzovax across the six timepoints; 1 = pre-vaccination, 2 = 14 days post vaccination, 3 = 35 days post vaccination, 4 = pre-challenge, 5 = 35 days post challenge and 6 = 3 weeks post lambing. Ewes were vaccinated on day 0 and subsequently challenged at day 90 of gestation with 1 × 10 IFU of C. abortus C95/27. Lambing occurred from day 125 of gestation until day 145 gestation. Dashed line indicates point of sero-positivity > 30 S/P% O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 4 of 9 Fig. 2 Tris-buffer ewes tested with MVD-Enfer kit. MVD-Enfer kit showing antibody levels in ewes vaccinated with Tris-buffer across the six timepoints; as described in Fig. 1. Vaccination, challenge and lambing of ewes occurred as described in Fig. 1. Dashed line indicates point of sero-positivity > 30 S/P% positive at both 35 days post challenge and at 3 weeks of 19.43, 14.62 and 3.79 respectively. These ewes also post lambing. Ewe 3551 tested doubtful at 35 days post tested negative at 35 days post vaccination, with an S/P% challenge with an S/P% of 56.09, but subsequently tested of 0.93, 5.20 and − 13.73 respectively. As regards the positive at 3 weeks post lambing. However, ewe 3407 challenge period, there was a general trend of rising anti- remained negative throughout the study; this ewe did body levels following inoculation. One ewe, 4773, tested not seroconvert at either 35 days post challenge or at negative at 3 weeks post lambing with an S/P% of 21.88, 3 weeks post lambing. The S/P% was 4.55 and 5.10 at but this ewe had tested positive previously at 35 days these time-points respectively. post challenge. The individual antibody levels for the Enzovax ewes Results for the Tris-buffer ewes using the LSI kit are recorded using the LSI kit are shown in Fig. 5. In gen- shown in Fig. 6. All ewes tested negative in the vaccine eral, antibody levels increased after vaccination. How- phase. Pre-challenge, all ewes in the group tested nega- ever, three ewes in this group tested negative at both 14 tive apart from two ewes, 3547 and 3559. These ewes and 35 days post vaccination. Ewes 4749, 4773 and 4901 had an S/P% of 36.56 and 46.41 respectively. At 35 days tested negative at day 14 post vaccination, with an S/P% post challenge, all ewes tested positive apart from one Fig. 3 Enzovax ewes tested with IDvet kit. IDvet kit showing antibody levels in ewes vaccinated with Enzovax across the six timepoints; as described in Fig. 1. Vaccination, challenge and lambing of ewes occurred as described in Fig. 1. Dashed line indicates point of sero-positivity > 60 S/P% O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 5 of 9 Fig. 4 Tris-buffer ewes tested with IDvet kit. IDvet kit showing antibody levels in ewes vaccinated with Tris-buffer across the six timepoints; as described in Fig. 1. Vaccination, challenge and lambing of ewes occurred as described in Fig. 1. Dashed line indicates point of sero-positivity > 60 S/P% ewe, 3538, which tested negative with an S/P% of 13.40. post challenge in both ewes that aborted and ewes that This ewe did test positive at 3 weeks post lambing, as had live lambs. The MVD-Enfer kit detected a statisti- did all other ewes in the group. cally significant difference between antibody levels in In an effort to determine if there was a correlation ewes at 3 weeks post lambing that had live or dead between antibody levels and whether or not a ewe would lambs (P value = 0.0482). Statistically significant results abort, graphs were constructed to examine the levels of were obtained with all kits with higher antibody levels at antibodies recorded from each ELISA in ewes that 3 weeks post lambing compared to 35 days post chal- aborted compared to ewes that had live lambs. All ten lenge for Tris-buffer control ewes (IDvet P = 0.0062, Enzovax vaccinated ewes had live lambs while only three MVD-Enfer P = 0.0021, LSI P = 0.0222). out of the nine Tris-buffer control ewes had live lambs The sensitivity of each ELISA kit was calculated (3258, 3547 and 4785). As shown in Fig. 7, antibody according to the Se equation described previously. The levels were higher at day 35 post challenge in ewes that IDvet kit had a sensitivity of 73.68% and the MVD-Enfer went on to abort compared to ewes that produced live kit had a sensitivity of 78.95%. The LSI kit had the high- lambs for all kits. Furthermore, antibody levels were est sensitivity, at 94.74%. The kappa coefficients demon- higher again at 3 weeks post lambing than at 35 days strated that all ELISAs had a “moderate to substantial” Fig. 5 Enzovax ewes tested with LSI kit. LSI kit showing antibody levels in ewes vaccinated with Enzovax across the six timepoints; as described in Fig. 1. Vaccination, challenge and lambing of ewes occurred as described in Fig. 1. Dashed line indicates point of sero-positivity > 25 S/P% O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 6 of 9 Fig. 6 Tris-buffer ewes tested with LSI kit. LSI kit showing antibody levels in ewes vaccinated with Tris-buffer across the six timepoints; as described in Fig. 1. Vaccination, challenge and lambing of ewes occurred as described in Fig. 1. Dashed line indicates point of sero-positivity > 25 S/P% agreement [14] with kappa values ranging from 0.59–0. and the ELISAs tested. In contrast, Longbottom et al. [11] 70 when calculated using sera from all time points. and Markey et al. [20]reported that ELISAsareamore Moderate agreement ranges from 0.41–0.60 while sensitive serological test than CFT. As knowledge of the substantial agreement ranges from 0.61–0.80 [14]. The outer membrane proteins of C. abortus has increased over percentage concordance between all three ELISAs was time, new antigenic proteins have been explored and have greater than 78% as shown in Table 1. resulted in the creation of novel ELISA plates [4, 11]. The aim of this current study was to compare three Discussion commercial ELISAs coated with three different antigens; Various studies have been conducted to date evaluating MOMP, POMP and LPS. All ELISAs demonstrated a the sensitivity and specificity of ELISAs compared to CFT “moderate to substantial” agreement (0.59–0.70) [14] in the diagnosis of C. abortus infection [9, 11, 17–20]. The using the kappa coefficient test while the percentage results of these studies vary, with McCauley et al. [18]de- of concordance was also greater than 78% in compar- scribing equal sensitivities and specificities between CFT ing all three ELISAs [16]. The LSI ELISA coated with Fig. 7 Antibody levels in ewes that had live lambs versus dead lambs. Comparison of antibody levels at 35 days post challenge and 3 weeks post lambing (~ 70 days post challenge) in ewes that had live or dead lambs. * marks a statistically significant difference between antibody levels in ewes at 3 weeks post lambing that had live or dead lambs (P value = 0.0482) O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 7 of 9 Table 1 Kappa coefficient values and concordance percentages Although an LPS-based ELISA fails to distinguish be- for comparing the LSI kit to MVD kits, LSI kit to ID vet kit and ID tween C. abortus and C. pecorum infected ewes, Griffiths vet kit to MVD kit using all data from the 6 time points; pre- et al. [22] suggest it is a more sensitive screening test vaccination, 14 days post vaccination, 35 days post vaccination, than the CFT, especially in flocks where abortion has pre-challenge, 35 days post challenge and 3 weeks post occurred. Kaltenboeck et al. [23] also showed that a lambing chlamydial LPS ELISA was more sensitive than the CFT MVD ELISA ID VET and may be a suitable replacement. Furthermore, a puri- LSI ELISA Kappa (κ) = 0.5887% Kappa (κ) = 0.6842% fied LPS-based ELISA prepared by Sting and Hafez [24] concordance = 78.9 concordance = 80.7 was shown to be more sensitive than the CFT. MVD ELISA Kappa (κ) = 0.7012% As regards MOMP-based ELISAs, much of the litera- concordance = 80.7 ture has examined their sensitivities in comparison to the CFT [4, 18, 25, 26]. A competitive ELISA described chlamydial LPS was the most sensitive at 94.74%, by Salti-Montesanto et al. [25] diagnosed 9 out of 10 in- when calculated at 3 weeks post lambing. This indi- fected flocks whereas the CFT detected only 6 out of 10 cates that the LSI kit found more animals to be posi- infected flocks in the study. Hoelzle et al. [26] suggested tive than the other kits and appears to be most that recombinant MOMP antigens are suitable for use in effective in diagnosing the infection in individual serological tests and may eliminate binding of cross- animals. As well as sensitivity, specificity rates are reactive antibodies. While McCauley et al. [18] reported essential data in determining the most effective sero- a sensitivity of 70.4% when using MOMP synthetic logical test. Buendía et al. [21] suggested that mixed peptide-based ELISA, other literature suggests that a infections of C. abortus and C. pecorum are common MOMP ELISA based on the variable segment 2 region in flocks. However, the specificities of these ELISAs yields similar results to the CFT, lacks sensitivity and is were not calculated in this study as sera from ewes therefore not suitable for use as a screening test [4]. known to be infected with other chlamydial species Numerous studies have investigated the use of POMP such as C. pecorum were not available. antigens in serological assays for the detection of C. Another limitation of this study was that the small abortus antibodies [9, 11, 19, 21, 27]. Buendía et al. [21] sample size with only 19 ewes included in the study. A reported a sensitivity of 90.9% when using an 80–90 kDa larger sample size would be beneficial in evaluating these POMP-based ELISA in comparison to the CFT which ELISAs and determining their sensitivities. Field sera was 71% sensitive, while also offering the possibility of from sheep naturally infected with C. abortus as well as differentiating rMOMP-vaccinated ewes from naturally sera from sheep known to be positive for C. pecorum infected ewes. In another study investigating a recom- would have been useful in evaluating these ELISAs. This binant POMP80–90 ELISA a sensitivity of 80% was cal- would also have allowed specificity data to be calculated. culated, which was higher in comparison to the CFT With the IDvet and MVD-Enfer kits in particular, a [19]. However, when using field sera, a lower sensitivity number of samples had to be retested as unexpected was obtained [19]. Longbottom et al. [27] described an results were obtained the first time they were tested. As indirect ELISA created using a recombinant POMP91B mentioned in the results, some ewes in the Enzovax fragment which yielded a sensitivity of 84.2%, which was group did not test positive at any time-point with the greater than that of the CFT. The ELISA also proved MVD-Enfer kit, even after retesting. This was an inter- more effective at distinguishing between C. abortus and esting finding and warrants further investigation. C. pecorum infections [27]. A further study was carried With the IDvet kit two Enzovax ewes remained nega- out based on a recombinant POMP90 ELISA [11]. Both tive after vaccination and challenge, even after the the rOMP90–3 and rOMP90–4 ELISAs had higher sen- samples were retested. These examples of sera that sitivities than the CFT, with the rOMP90–4 fragment required retesting show that the accuracy of these tests being more sensitive than the rOMP90–3 fragment must be taken into consideration and demonstrates that when testing field sera [11]. In contrast, Wilson et al. [9] the results were sometimes difficult to interpret. The LSI reported the rOMP90–3 ELISA as being more sensitive kit therefore seemed to be the most effective kit in this than both the CFT and the rOMP90–4 ELISA, with re- study but a greater number of samples would need to sults of 96.8, 93.5 and 91.9% respectively. A sensitivity of be tested across more time-points to confirm this. One 96.8% with the rOMP90–3 ELISA [9] is higher than the possible option would be to screen flocks with the LSI result of 78.95% obtained in the current study. kit to identify all ewes infected with Chlamydia species Interestingly anti-chlamydial antibody levels were found and then to use the other kits to detect ewes infected to be consistently higher after challenge in ewes that with C. abortus only. However, this approach may not aborted compared to ewes that had live lambs. Studies be economically feasible. have shown increases in antibody levels associated with C. O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 8 of 9 abortus reproductive failure [28–30]. Gutierrez et al. [31] higher at 3 weeks post lambing than at 35 days post and García-Seco et al. [32] have also noted that antibody challenge. Future work involving more sera, as well as titres were lower in animals that did not go on to abort. testing at more time-points, would be beneficial in This current study and others clearly show how elevated confirming the results obtained in this study. antibody levels could form a basis to identify ewes that Acknowledgements will go on to abort. The antibody increase observed in all The authors gratefully acknowledge the support of the Thomas O’Hanlon groups at 3 weeks post lambing could be associated with Bursary for AOD. Additionally the authors gratefully acknowledge the research support of Teagasc, UCD and DAFM. the development of protective immunity against subse- quent abortions as described by García-Seco et al. [32] Funding and Longbottom and Coulter [33]. Alternatively it may The authors gratefully acknowledge the funding support for the project from the Thomas O’Hanlon Bursary for AOD and from the School of Veterinary simply be a diagnostic marker of exposure to high num- Medicine, UCD for laboratory consumables. Additionally the authors bers of circulating C. abortus. In any case early warning of gratefully acknowledge the funding support of Teagasc for LMON and of the impending abortions could have significant benefits in Department of Agriculture, Food and the Marine (DAFM) in carrying out the experimental trial. terms of targeting long- acting antimicrobial treat- ment [3] towards those animals in imminent danger Availability of data and materials of abortion rather than having to administer treat- The data sets used and analysed during the current study are available from the corresponding author on reasonable request. ment to the whole flock. It is clear from the literature available that a lot of Authors’ contributions variation exists in the sensitivities of different ELISAs. In LMON and BM collected the serum for this study. AOD and LMON ran the ELISAs and analysed the resulting data. LMON, AOD and BM contributed in general, ELISAs have been shown to be more sensitive writing this manuscript. All authors read and approved the final manuscript. than CFT, in particular the POMP ELISAs as described above. Hagemann et al. [34] described how antibodies to Ethics approval and consent to participate Ethical approval was granted by the Animal Research Ethics Committee virulence-associated antigens such as chlamydial protease- (AREC) at University College Dublin (UCD) under license AREC-14-10 Markey like activity factor (CPAF) are not present for more than and all procedures were performed under experimental license from the 18 weeks after abortion in most cases, whereas antibodies Health Products Regulatory Authority (HPRA) for Ireland under license AE 19113/P004. to surface antigens such as MOMP persist for longer. However, in ewes that lamb normally, antibodies to sur- Competing interests face antigens do not persist [34]. It has also been shown The authors declare that they have no competing interests. by Livingstone et al. [4] that anti-MOMP antibodies only increase around the time of abortion and then gradually Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in decline, while anti-POMP antibodies appear earlier and published maps and institutional affiliations. may persist for longer. It is therefore evident that differ- ences in immunoreactivites may be due to the time-points Received: 13 October 2017 Accepted: 10 May 2018 at which blood samples are taken following abortion, as described by Forsbach-birk et al. [35]. It is important to References note that the current study was limited to six specific 1. All-Island Animal Disease Surveillance Report 2015 https://www.agriculture. gov.ie/media/migration/animalhealthwelfare/labservice/rvlreports/2016/ time-points and therefore the peak level of antibody, after 2015AFBIDAFMAllIslandSurveillanceReport190117.pdf. abortion may have been missed. Future work would be 2. Wong S, Gray E, Buxton D, Finlayson J, Johnson F. 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Epitope mapping with solid-phase peptides: 30. Papp JR, Shewen PE, Gartley CJ. Abortion and subsequent excretion of identification of type-, subspecies-, species- and genus-reactive antibody chlamydiae from the reproductive tract of sheep during estrus. Infect binding domains on the major outer membrane protein of Chlamydia Immun. 1994;62(9):3786–92. trachomatis. Mol Microbiol. 1988;2(5):673–9. 31. Gutierrez J, Williams E, O’Donovan J, Brady C, Proctor A, Marques P, Worrall 11. Longbottom D, Fairley S, Chapman S, Psarrou E, Vretou E, Livingstone M. S, Nally J, McElroy M, Bassett H, Sammin D, Markey B. Monitoring clinical Serological diagnosis of ovine enzootic abortion by enzyme-linked outcomes, pathological changes and shedding of Chlamydophila abortus immunosorbent assay with a recombinant protein fragment of the following experimental challenge of periparturient ewes utilizing the natural polymorphic outer membrane protein POMP90 of Chlamydophila abortus.J route of infection. 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Vet factors and candidates for Serodiagnosis. PLoS One. 2013;8(11):80310. Rec. 1997;141(7):164–8. 18. McCauley L, Lancaster M, Young P, Butler K, Ainsworth C. Comparison of ELISA and CFT assays for Chlamydophila abortus antibodies in ovine sera. Aust Vet J. 2007;85(8):325–8. 19. Vretou E, Radouani F, Psarrou E, Kritikos I, Xylouri E, Mangana O. Evaluation of two commercial assays for the detection of Chlamydophila abortus antibodies. Vet Microbiol. 2007;123(1–3):153–61. 20. Markey B, McNulty M, Todd D. Comparison of serological tests for the diagnosis of Chlamydia psittaci infection of sheep. Vet Microbiol. 1993; 36(3–4):233–52. 21. Buend́ ıa A, Cuello F, Del Rio L, Gallego M, Caro M, Salinas J. Field evaluation of a new commercially available ELISA based on a recombinant antigen for diagnosing Chlamydophila abortus (Chlamydia psittaci serotype 1) infection. Vet Microbiol. 2001;78(3):229–39. 22. Griffiths PC, Plater JM, Horigan MW, Rose MP, Venables C, Dawson M. Serological diagnosis of ovine enzootic abortion by comparative inclusion immunofluorescence assay, recombinant lipopolysaccharide enzyme-linked immunosorbent assay, and complement fixation test. J Clin Microbiol. 1996; 34(6):1512–8. 23. Kaltenboeck B, Heard D, DeGraves FJ, Schmeer N. Use of synthetic antigens improves detection by enzyme-linked immunosorbent assay of antibodies against abortigenic Chlamydia psittaci in ruminants. J Clin Microbiol. 1997; 35(9):2293–8. 24. Sting R, Hafez H. Purification of Chlamydia psittaci antigen by affinity chromatography on polymyxin B agarose for use in the enzyme- linked immunosorbent assay (ELISA). Zentralblatt für Bakteriologie. 1992;277(4):436–45. 25. Salti-Montesanto V, Tsoli E, Papavassiliou P, Psarrou E, Markey BK, Jones GE, Vretou E. Diagnosis of ovine enzootic abortion, using a competitive ELISA based on monoclonal antibodies against variable segments 1 and 2 of the major outer membrane protein of Chlamydia psittaci serotype 1. Am J Vet Res. 1997;58(3):228–35. 26. Hoelzle L, Hoelzle K, Wittenbrink M. Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera. Vet Microbiol. 2004;103(1–2):85–90. 27. Longbottom D, Psarrou E, Livingstone M, Vretou E. Diagnosis of ovine enzootic abortion using an indirect ELISA (rOMP91B iELISA) based on a recombinant protein fragment of the polymorphic outer membrane protein POMP91B of Chlamydophila abortus. FEMS Microbiol Lett. 2001;195(2):157–61. 28. Wilsmore A, Wilsmore B, Dagnall G, Izzard K, Woodland R, Dawson M, Venables C. Clinical and immunological responses of ewes following vaccination with an experimental formalin-inactivated Chlamydia psittaci http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Irish Veterinary Journal Springer Journals

Comparison of three commercial serological tests for the detection of Chlamydia abortus infection in ewes

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Abstract

Background: 20.9% of diagnosable abortions in Ireland in 2015 were caused by Chlamydia abortus infection. Abortion usually occurs in the last 2–3 weeks of gestation, and up to 30% of ewes may be affected in naïve flocks. Serological diagnosis of EAE in flocks using LPS or whole bacteria as antigens is often hindered by cross reactions with C. pecorum. Although the complement fixation test is the official test for diagnosis of EAE, more sensitive and specific ELISA based tests have been developed. This study aimed to compare three commercial ELISA kits to detect C. abortus antibodies in ewes and to determine which of the kits had the highest sensitivity. The IDvet kit utilises a MOMP peptide antigen, the MVD-Enfer kit is based on a POMP90–3 antigen while the LSI kit plates are coated with chlamydial LPS. The study also aimed to examine the potential of these ELISAs to distinguish infected animals that go on to abort compared to those that have live lambs. Ewes were vaccinated with either a commercial live vaccine (n = 10) or Tris-buffer sham inoculation (n = 9) 5 months prior to gestation, these ewes were then challenged with C. abortus (1 × 10 IFU/ml) on day 90 of gestation. Sera were collected at pre- vaccination, 14 days post vaccination, 35 days post vaccination, pre-challenge, 35 days post challenge and 3 weeks post lambing/abortion (~ 70 days post challenge) and tested using the 3 aforementioned ELISAs to determine if one ELISA was more sensitive at detecting circulating anti-chlamydial antibodies. Results: Sensitivity was highest with the LSI test kit at 94.74%, followed by the MVD-Enfer and IDvet kits, at 78.95 and 73.68% respectively. Ewes vaccinated with Enzovax became seropositive at 14 days post vaccination with all kits. Following challenge at day 90 of gestation, antibody titres steadily rose in all groups of ewes. With all ELISA kits, antibody levels were higher in ewes that aborted compared to ewes that had live lambs at 35 days post challenge and three weeks post lambing, and statistically significantly higher antibody levels were recorded in ewes that aborted compared to ewes that had live lambs using the MVD-ENFER ELISA at three weeks post lambing (P = 0.0482). Conclusions: The LSI assay was the most sensitive out of the three kits tested in this study, when sera were tested at three weeks post lambing. As the LPS used in this kit is cross-reactive with all chlamydia, it is good for identifying flocks infected with any chlamydial species, but it is not considered specific for C. abortus. Furthermore, antibody levels were higher in ewes that aborted compared to ewes that had live lambs, at both 35 days post challenge and at three weeks post lambing. Future work should include evaluation of a larger number of sera at a wider range of time-points as well as an estimation of the specificity of commercially available assays. Keywords: Chlamydia abortus, Enzootic abortion of ewes, ELISA sensitivity, ELISA comparison * Correspondence: lauren.oneill@ucd.ie L. M. O’Neill and Á. O’Driscoll contributed equally to this work. School of Veterinary Medicine, University College Dublin, Room 07A, Belfield, Dublin 4, Ireland © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 2 of 9 Background potential of the different kits to identify ewes that subse- Enzootic abortion of ewes (EAE) is the second most quently progress to abortion. common infectious cause of ovine abortion in Ireland, causing 20.9% of diagnosable abortions in 2015 [1]. The Method causative agent, Chlamydia abortus, is a serious zoonotic Sera from two groups of ewes (n = 19) were tested using pathogen in pregnant women which makes accurate three different commercial ELISA kits: IDvet, LSI and testing essential in the diagnosis and control of infected MVD-Enfer. The sera were generated as part of a sheep [2]. The disease manifests itself as a placentitis, vaccination trial [13]. This trial involved 5 different leading to abortion in the last 2 to 3 weeks of gestation treatment groups including the testing of novel recom- [3]. Aborting ewes are the principal means of transmis- binant protein vaccines against EAE. The sera from the sion of infection but rarely show prior clinical signs. As positive control ewes that received a live commercial a result early, detection of infection during pregnancy is vaccine and the negative control ewes that received a vital in permitting therapeutic intervention and control- sham Tris-buffer inoculation were used in the current ling the spread of disease. study. In summary, ten ewes were vaccinated 5 months Serological diagnosis of EAE in flocks is challenging prior to mating with Enzovax (MSD Animal Health), the for a number of reasons. When serological tests such as commercial vaccine used in Ireland, while the nine were the complement fixation test (CFT) use LPS or whole given a sham inoculation of Tris-buffer (control ewes). bacteria as antigens in the diagnosis of C. abortus infec- All the ewes were challenged by subcutaneous inoculation tion, specificity tends to be low as cross reactions with at day 90 of gestation with 10 IFU of C. abortus isolate Chlamydia pecorum are often observed [4]. Chlamydia C95/27. All sera were tested at six time-points: 1 = pre- pecorum mainly causes inapparent enteric infections [5], vaccination, 2 = 14 days post vaccination, 3 = 35 days post but is also known to cause arthritis, conjunctivitis and vaccination, 4 = pre-challenge, 5 = 35 days post challenge pneumonia [3]. Cross-reactive antibodies may arise from and 6 = three weeks post lambing. All sera were thawed at other chlamydial species and certain Gram-negative room temperature while the kit reagents were brought to bacteria such as Acinetobacter spp. [4]. Thus, specific room temperature. The ELISAs were carried out accord- diagnostic tests are required to properly understand the ing to manufacturers’ instructions. The format of each epidemiology of EAE and in implementing control ELISA tested was an indirect ELISA. The S/P% was calcu- strategies. The diagnosis of EAE is further complicated lated for each sample to determine the titres of antibodies by the ability of C. abortus to cause latent infections [6]. using the formula [OD -OD ]/ [OD sample negative control positive Pathological lesions in the ovine placenta do not usually -OD ] × 100. For the LSI kit, a titre >25 control negative control develop until day 90 of gestation [7]. S/P% was considered positive. For the IDvet kit, an S/P% The CFT is the most widely used technique for ≤50% was considered negative, an S/P% greater than 50% serological diagnosis of EAE and is considered the gold and less than 60% was considered doubtful and an S/ standard test for official trade purposes [8]. However, P%≥60% was considered positive. For the MVD-Enfer kit, advances have been made in the development of more an S/P% ≤20% was considered negative, an S/P% greater sensitive and specific tests such as ELISAs that specific- than 20% and less than 30% was considered doubtful and ally detect antibodies to a range of chlamydial antigens an S/P% ≥30%was considered positive. including major outer membrane protein (MOMP) and polymorphic outer membrane proteins (POMP) [9]. Statistical analysis The aim of this study was to compare three commer- Statistical analysis of results was conducted on Graph- cial ELISAs designed to detect antibodies to C. abortus Pad Prism 7.03. Parametric unpaired t tests were carried in ewes. Each ELISA utilises a different chlamydial anti- out. Sensitivities (Se) of the kits were calculated using gen. In the IDvet kit, wells are coated with a MOMP the results from three weeks post lambing and the fol- peptide antigen, which is known to have genus-, species- lowing formula: , subspecies- and serotype-specific immunodomains The number of diseased animals testing positive [10]. Wells of the MVD-Enfer kit are coated with a Se ¼ The total number of diseased animals POMP90–3 antigen considered to be specific for C. abortus [11]. The LSI kit uses chlamydial LPS as the The agreement between the ELISAs was determined antigen coating the wells. This is a genus-specific anti- from the Kappa coefficient (κ) and percentage concord- gen common to all chlamydial species [12]. ance for samples from all time points [14] using Epi- The study aimed to determine which of the three kits Tools epidemiological calculators [15]. Concordance was had the highest sensitivity and to evaluate the influence calculated as the sum of positive –positive values and of the different antigens used in the assays on test char- negative-negative values expressed as a percentage of the acteristics. Furthermore, the study aimed to evaluate the total number of serum samples [16]. O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 3 of 9 Results following challenge. Ewes 3376, 3554 and 4901 had an All ELISA kits detected a similar trend in antibody levels S/P% of 1.18, 1.34 and 6.22 respectively, at 3 weeks post post-vaccination and post-challenge. The antibody levels lambing. For the other ewes there was an increase in in the two groups of ewes at the six time-points are dis- antibody levels after vaccination and again after chal- played in Figs. 1, 2, 3, 4, 5 and 6. The ewes vaccinated lenge at day 90 of gestation. with the commercial vaccine showed a rise in antibody The results of the MVD-Enfer kit for each individual titre after vaccination (Figs. 1, 3 and 5). This is to be Tris-buffer ewe are shown in Fig. 2. As expected, all expected as it is a live vaccine and the animals were ewes tested negative following the sham inoculation. At therefore exposed to the full range of chlamydial anti- 35 days post challenge some ewes begin to test positive, gens. Antibody levels in both groups rose after challenge with all ewes in the group testing positive at 3 weeks at day 90 of gestation (Figs. 1, 2, 3, 4, 5 and 6). post-lambing. Ewe 3547 tested doubtful at 35 days post All ewes showed a steady rise in antibody levels after challenge with an S/P% of 24.48, but this ewe subse- challenge, however the Enzovax vaccinated ewes at day quently tested positive at 3 weeks post lambing. 35 post challenge appeared to plateau (Figs. 1 and 3) The antibody levels in individual ewes from the Enzo- unlike Tris-buffer vaccinated ewes (Figs. 2 and 4). It is vax group using the IDvet kit are shown in Fig. 3.In important to note that in general the IDvet kit recorded general, antibody levels rose following vaccination. How- higher antibody levels for both vaccine groups and ever, four ewes did not test positive at either 14 or peaked at levels higher than 150 S/P% (Fig. 3). The other 35 days post vaccination. Ewe 3376 tested doubtful at ELISA kits did not exceed 150 S/P% (Figs. 1 and 5). day 14 with an S/P% of 51.17 and tested negative at day All Enzovax ewes after vaccination demonstrated a 35 with an S/P% of 25.19. Ewe 3554 tested negative at peak similar to that post-challenge, showing the diffi- day 14 and day 35, with an S/P% of 23.33 and 19.18 re- culty of differentiating vaccinated ewes from infected spectively. Ewe 4773 also tested negative at day 14 and ewes using the current commercial vaccine and available day 35, with an S/P% of 49.81 and 25.21 respectively. ELISAs. Ewe 4901 tested doubtful at day 14 with an S/P% of 56. The results of the MVD-Enfer kit for each individual 90 and tested negative at day 35 with an S/P% of 17.31. Enzovax vaccinated ewe are detailed in Fig. 1. At day 14 Following challenge, there was a general rise in antibody post vaccination, four ewes tested negative and one ewe levels, although four ewes did not fit this trend. At tested doubtful. Ewe number 3376 tested doubtful with 3 weeks post lambing, ewe 3543 tested doubtful, with an an S/P% of 26.69. Ewes 3543, 3554, 4773, 4901 all tested S/P% of 53.01. Furthermore, ewes 3554, 4749 and 4773 negative, with an S/P% of 6.39, 2.56, 12.23 and 15.66 re- all tested negative at 3 weeks post lambing, with an spectively. These five ewes all tested negative at day 35 S/P% of 9.04, 25.49 and 8.29 respectively. post vaccination. Three ewes tested negative at both The results of the IDvet kit for Tris-buffer ewes are 35 days post challenge and 3 weeks post lambing, so shown in Fig. 4. All ewes tested negative in the vaccine antibody levels of these ewes did not show an increase phase of the study. Most ewes in the group tested Fig. 1 Enzovax ewes tested with MVD-Enfer ki. MVD-Enfer kit showing antibody levels in ewes vaccinated with Enzovax across the six timepoints; 1 = pre-vaccination, 2 = 14 days post vaccination, 3 = 35 days post vaccination, 4 = pre-challenge, 5 = 35 days post challenge and 6 = 3 weeks post lambing. Ewes were vaccinated on day 0 and subsequently challenged at day 90 of gestation with 1 × 10 IFU of C. abortus C95/27. Lambing occurred from day 125 of gestation until day 145 gestation. Dashed line indicates point of sero-positivity > 30 S/P% O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 4 of 9 Fig. 2 Tris-buffer ewes tested with MVD-Enfer kit. MVD-Enfer kit showing antibody levels in ewes vaccinated with Tris-buffer across the six timepoints; as described in Fig. 1. Vaccination, challenge and lambing of ewes occurred as described in Fig. 1. Dashed line indicates point of sero-positivity > 30 S/P% positive at both 35 days post challenge and at 3 weeks of 19.43, 14.62 and 3.79 respectively. These ewes also post lambing. Ewe 3551 tested doubtful at 35 days post tested negative at 35 days post vaccination, with an S/P% challenge with an S/P% of 56.09, but subsequently tested of 0.93, 5.20 and − 13.73 respectively. As regards the positive at 3 weeks post lambing. However, ewe 3407 challenge period, there was a general trend of rising anti- remained negative throughout the study; this ewe did body levels following inoculation. One ewe, 4773, tested not seroconvert at either 35 days post challenge or at negative at 3 weeks post lambing with an S/P% of 21.88, 3 weeks post lambing. The S/P% was 4.55 and 5.10 at but this ewe had tested positive previously at 35 days these time-points respectively. post challenge. The individual antibody levels for the Enzovax ewes Results for the Tris-buffer ewes using the LSI kit are recorded using the LSI kit are shown in Fig. 5. In gen- shown in Fig. 6. All ewes tested negative in the vaccine eral, antibody levels increased after vaccination. How- phase. Pre-challenge, all ewes in the group tested nega- ever, three ewes in this group tested negative at both 14 tive apart from two ewes, 3547 and 3559. These ewes and 35 days post vaccination. Ewes 4749, 4773 and 4901 had an S/P% of 36.56 and 46.41 respectively. At 35 days tested negative at day 14 post vaccination, with an S/P% post challenge, all ewes tested positive apart from one Fig. 3 Enzovax ewes tested with IDvet kit. IDvet kit showing antibody levels in ewes vaccinated with Enzovax across the six timepoints; as described in Fig. 1. Vaccination, challenge and lambing of ewes occurred as described in Fig. 1. Dashed line indicates point of sero-positivity > 60 S/P% O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 5 of 9 Fig. 4 Tris-buffer ewes tested with IDvet kit. IDvet kit showing antibody levels in ewes vaccinated with Tris-buffer across the six timepoints; as described in Fig. 1. Vaccination, challenge and lambing of ewes occurred as described in Fig. 1. Dashed line indicates point of sero-positivity > 60 S/P% ewe, 3538, which tested negative with an S/P% of 13.40. post challenge in both ewes that aborted and ewes that This ewe did test positive at 3 weeks post lambing, as had live lambs. The MVD-Enfer kit detected a statisti- did all other ewes in the group. cally significant difference between antibody levels in In an effort to determine if there was a correlation ewes at 3 weeks post lambing that had live or dead between antibody levels and whether or not a ewe would lambs (P value = 0.0482). Statistically significant results abort, graphs were constructed to examine the levels of were obtained with all kits with higher antibody levels at antibodies recorded from each ELISA in ewes that 3 weeks post lambing compared to 35 days post chal- aborted compared to ewes that had live lambs. All ten lenge for Tris-buffer control ewes (IDvet P = 0.0062, Enzovax vaccinated ewes had live lambs while only three MVD-Enfer P = 0.0021, LSI P = 0.0222). out of the nine Tris-buffer control ewes had live lambs The sensitivity of each ELISA kit was calculated (3258, 3547 and 4785). As shown in Fig. 7, antibody according to the Se equation described previously. The levels were higher at day 35 post challenge in ewes that IDvet kit had a sensitivity of 73.68% and the MVD-Enfer went on to abort compared to ewes that produced live kit had a sensitivity of 78.95%. The LSI kit had the high- lambs for all kits. Furthermore, antibody levels were est sensitivity, at 94.74%. The kappa coefficients demon- higher again at 3 weeks post lambing than at 35 days strated that all ELISAs had a “moderate to substantial” Fig. 5 Enzovax ewes tested with LSI kit. LSI kit showing antibody levels in ewes vaccinated with Enzovax across the six timepoints; as described in Fig. 1. Vaccination, challenge and lambing of ewes occurred as described in Fig. 1. Dashed line indicates point of sero-positivity > 25 S/P% O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 6 of 9 Fig. 6 Tris-buffer ewes tested with LSI kit. LSI kit showing antibody levels in ewes vaccinated with Tris-buffer across the six timepoints; as described in Fig. 1. Vaccination, challenge and lambing of ewes occurred as described in Fig. 1. Dashed line indicates point of sero-positivity > 25 S/P% agreement [14] with kappa values ranging from 0.59–0. and the ELISAs tested. In contrast, Longbottom et al. [11] 70 when calculated using sera from all time points. and Markey et al. [20]reported that ELISAsareamore Moderate agreement ranges from 0.41–0.60 while sensitive serological test than CFT. As knowledge of the substantial agreement ranges from 0.61–0.80 [14]. The outer membrane proteins of C. abortus has increased over percentage concordance between all three ELISAs was time, new antigenic proteins have been explored and have greater than 78% as shown in Table 1. resulted in the creation of novel ELISA plates [4, 11]. The aim of this current study was to compare three Discussion commercial ELISAs coated with three different antigens; Various studies have been conducted to date evaluating MOMP, POMP and LPS. All ELISAs demonstrated a the sensitivity and specificity of ELISAs compared to CFT “moderate to substantial” agreement (0.59–0.70) [14] in the diagnosis of C. abortus infection [9, 11, 17–20]. The using the kappa coefficient test while the percentage results of these studies vary, with McCauley et al. [18]de- of concordance was also greater than 78% in compar- scribing equal sensitivities and specificities between CFT ing all three ELISAs [16]. The LSI ELISA coated with Fig. 7 Antibody levels in ewes that had live lambs versus dead lambs. Comparison of antibody levels at 35 days post challenge and 3 weeks post lambing (~ 70 days post challenge) in ewes that had live or dead lambs. * marks a statistically significant difference between antibody levels in ewes at 3 weeks post lambing that had live or dead lambs (P value = 0.0482) O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 7 of 9 Table 1 Kappa coefficient values and concordance percentages Although an LPS-based ELISA fails to distinguish be- for comparing the LSI kit to MVD kits, LSI kit to ID vet kit and ID tween C. abortus and C. pecorum infected ewes, Griffiths vet kit to MVD kit using all data from the 6 time points; pre- et al. [22] suggest it is a more sensitive screening test vaccination, 14 days post vaccination, 35 days post vaccination, than the CFT, especially in flocks where abortion has pre-challenge, 35 days post challenge and 3 weeks post occurred. Kaltenboeck et al. [23] also showed that a lambing chlamydial LPS ELISA was more sensitive than the CFT MVD ELISA ID VET and may be a suitable replacement. Furthermore, a puri- LSI ELISA Kappa (κ) = 0.5887% Kappa (κ) = 0.6842% fied LPS-based ELISA prepared by Sting and Hafez [24] concordance = 78.9 concordance = 80.7 was shown to be more sensitive than the CFT. MVD ELISA Kappa (κ) = 0.7012% As regards MOMP-based ELISAs, much of the litera- concordance = 80.7 ture has examined their sensitivities in comparison to the CFT [4, 18, 25, 26]. A competitive ELISA described chlamydial LPS was the most sensitive at 94.74%, by Salti-Montesanto et al. [25] diagnosed 9 out of 10 in- when calculated at 3 weeks post lambing. This indi- fected flocks whereas the CFT detected only 6 out of 10 cates that the LSI kit found more animals to be posi- infected flocks in the study. Hoelzle et al. [26] suggested tive than the other kits and appears to be most that recombinant MOMP antigens are suitable for use in effective in diagnosing the infection in individual serological tests and may eliminate binding of cross- animals. As well as sensitivity, specificity rates are reactive antibodies. While McCauley et al. [18] reported essential data in determining the most effective sero- a sensitivity of 70.4% when using MOMP synthetic logical test. Buendía et al. [21] suggested that mixed peptide-based ELISA, other literature suggests that a infections of C. abortus and C. pecorum are common MOMP ELISA based on the variable segment 2 region in flocks. However, the specificities of these ELISAs yields similar results to the CFT, lacks sensitivity and is were not calculated in this study as sera from ewes therefore not suitable for use as a screening test [4]. known to be infected with other chlamydial species Numerous studies have investigated the use of POMP such as C. pecorum were not available. antigens in serological assays for the detection of C. Another limitation of this study was that the small abortus antibodies [9, 11, 19, 21, 27]. Buendía et al. [21] sample size with only 19 ewes included in the study. A reported a sensitivity of 90.9% when using an 80–90 kDa larger sample size would be beneficial in evaluating these POMP-based ELISA in comparison to the CFT which ELISAs and determining their sensitivities. Field sera was 71% sensitive, while also offering the possibility of from sheep naturally infected with C. abortus as well as differentiating rMOMP-vaccinated ewes from naturally sera from sheep known to be positive for C. pecorum infected ewes. In another study investigating a recom- would have been useful in evaluating these ELISAs. This binant POMP80–90 ELISA a sensitivity of 80% was cal- would also have allowed specificity data to be calculated. culated, which was higher in comparison to the CFT With the IDvet and MVD-Enfer kits in particular, a [19]. However, when using field sera, a lower sensitivity number of samples had to be retested as unexpected was obtained [19]. Longbottom et al. [27] described an results were obtained the first time they were tested. As indirect ELISA created using a recombinant POMP91B mentioned in the results, some ewes in the Enzovax fragment which yielded a sensitivity of 84.2%, which was group did not test positive at any time-point with the greater than that of the CFT. The ELISA also proved MVD-Enfer kit, even after retesting. This was an inter- more effective at distinguishing between C. abortus and esting finding and warrants further investigation. C. pecorum infections [27]. A further study was carried With the IDvet kit two Enzovax ewes remained nega- out based on a recombinant POMP90 ELISA [11]. Both tive after vaccination and challenge, even after the the rOMP90–3 and rOMP90–4 ELISAs had higher sen- samples were retested. These examples of sera that sitivities than the CFT, with the rOMP90–4 fragment required retesting show that the accuracy of these tests being more sensitive than the rOMP90–3 fragment must be taken into consideration and demonstrates that when testing field sera [11]. In contrast, Wilson et al. [9] the results were sometimes difficult to interpret. The LSI reported the rOMP90–3 ELISA as being more sensitive kit therefore seemed to be the most effective kit in this than both the CFT and the rOMP90–4 ELISA, with re- study but a greater number of samples would need to sults of 96.8, 93.5 and 91.9% respectively. A sensitivity of be tested across more time-points to confirm this. One 96.8% with the rOMP90–3 ELISA [9] is higher than the possible option would be to screen flocks with the LSI result of 78.95% obtained in the current study. kit to identify all ewes infected with Chlamydia species Interestingly anti-chlamydial antibody levels were found and then to use the other kits to detect ewes infected to be consistently higher after challenge in ewes that with C. abortus only. However, this approach may not aborted compared to ewes that had live lambs. Studies be economically feasible. have shown increases in antibody levels associated with C. O’Neill et al. Irish Veterinary Journal (2018) 71:13 Page 8 of 9 abortus reproductive failure [28–30]. Gutierrez et al. [31] higher at 3 weeks post lambing than at 35 days post and García-Seco et al. [32] have also noted that antibody challenge. Future work involving more sera, as well as titres were lower in animals that did not go on to abort. testing at more time-points, would be beneficial in This current study and others clearly show how elevated confirming the results obtained in this study. antibody levels could form a basis to identify ewes that Acknowledgements will go on to abort. The antibody increase observed in all The authors gratefully acknowledge the support of the Thomas O’Hanlon groups at 3 weeks post lambing could be associated with Bursary for AOD. Additionally the authors gratefully acknowledge the research support of Teagasc, UCD and DAFM. the development of protective immunity against subse- quent abortions as described by García-Seco et al. [32] Funding and Longbottom and Coulter [33]. Alternatively it may The authors gratefully acknowledge the funding support for the project from the Thomas O’Hanlon Bursary for AOD and from the School of Veterinary simply be a diagnostic marker of exposure to high num- Medicine, UCD for laboratory consumables. Additionally the authors bers of circulating C. abortus. In any case early warning of gratefully acknowledge the funding support of Teagasc for LMON and of the impending abortions could have significant benefits in Department of Agriculture, Food and the Marine (DAFM) in carrying out the experimental trial. terms of targeting long- acting antimicrobial treat- ment [3] towards those animals in imminent danger Availability of data and materials of abortion rather than having to administer treat- The data sets used and analysed during the current study are available from the corresponding author on reasonable request. ment to the whole flock. It is clear from the literature available that a lot of Authors’ contributions variation exists in the sensitivities of different ELISAs. In LMON and BM collected the serum for this study. AOD and LMON ran the ELISAs and analysed the resulting data. LMON, AOD and BM contributed in general, ELISAs have been shown to be more sensitive writing this manuscript. All authors read and approved the final manuscript. than CFT, in particular the POMP ELISAs as described above. Hagemann et al. [34] described how antibodies to Ethics approval and consent to participate Ethical approval was granted by the Animal Research Ethics Committee virulence-associated antigens such as chlamydial protease- (AREC) at University College Dublin (UCD) under license AREC-14-10 Markey like activity factor (CPAF) are not present for more than and all procedures were performed under experimental license from the 18 weeks after abortion in most cases, whereas antibodies Health Products Regulatory Authority (HPRA) for Ireland under license AE 19113/P004. to surface antigens such as MOMP persist for longer. However, in ewes that lamb normally, antibodies to sur- Competing interests face antigens do not persist [34]. It has also been shown The authors declare that they have no competing interests. by Livingstone et al. [4] that anti-MOMP antibodies only increase around the time of abortion and then gradually Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in decline, while anti-POMP antibodies appear earlier and published maps and institutional affiliations. may persist for longer. It is therefore evident that differ- ences in immunoreactivites may be due to the time-points Received: 13 October 2017 Accepted: 10 May 2018 at which blood samples are taken following abortion, as described by Forsbach-birk et al. [35]. It is important to References note that the current study was limited to six specific 1. All-Island Animal Disease Surveillance Report 2015 https://www.agriculture. gov.ie/media/migration/animalhealthwelfare/labservice/rvlreports/2016/ time-points and therefore the peak level of antibody, after 2015AFBIDAFMAllIslandSurveillanceReport190117.pdf. abortion may have been missed. Future work would be 2. Wong S, Gray E, Buxton D, Finlayson J, Johnson F. Acute placentitis and valuable in assessing these ELISAs using sera from differ- spontaneous abortion caused by Chlamydia psittaci of sheep origin: a histological and ultrastructural study. J Clin Pathol. 1985;38(6):707–11. ent time-points, for example at various points throughout 3. Aitken I, Longbottom D. Chlamydial abortion. In: Diseases of sheep. 4th ed. gestation and beyond 3 weeks post lambing. Oxford: Blackwell Science; 2007. p. 105–12. 4. Livingstone M, Entrican G, Wattegedera S, Buxton D, McKendrick I, Longbottom D. Antibody responses to recombinant protein fragments of Conclusions the major outer membrane protein and polymorphic outer membrane This study demonstrated that when sera are tested at protein POMP90 in Chlamydophila abortus-infected pregnant sheep. Clin 3 weeks post lambing the LSI kit was the most sensitive Vaccine Immunol. 2005;12(6):770–7. 5. Berri M, Rekiki A, Boumedine K, Rodolakis A. Simultaneous differential of the three commercial kits examined. 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Irish Veterinary JournalSpringer Journals

Published: May 29, 2018

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