Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay
ABSTRACT
The obligate intracellular bacterium Chlamydia abortus is the causative agent of enzootic abortion of ewes and poses a significant zoonotic risk for pregnant women. Using proteomic analysis and gene expression library screening in a previous project, we identified potential virulence factors and candidates for serodiagnosis, of which nine were scrutinized here with a strip immunoassay. We have shown that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, Pmp13G) and virulence-associated (CPAF, TARP, SINC) antigens. While the latter disappeared within 18 weeks following abortion in a majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, nonaborting experimentally infected sheep developed mainly antibodies to surface antigens (MOMP, MIP, Pmp13G), all of which did not persist. We were also able to detect antibodies to these surface antigens in C. abortus-infected women who had undergone septic abortion, whereas a group of shepherds and veterinarians with occupational exposure to C. abortus-infected sheep revealed only sporadic immune responses to the antigens selected. The most specific antigen for the serodiagnosis of human C. abortus infections was Pmp13G, which showed no cross-reactivity with other chlamydiae infecting humans. We suggest that Pmp13G-based serodiagnosis accomplished by the detection of antibodies to virulence-associated antigens such as CPAF, TARP, and SINC may improve the laboratory diagnosis of human and animal C. abortus infections.
INTRODUCTION
Chlamydia abortus is an obligate intracellularly replicating zoonotic bacterium that shares a characteristic biphasic developmental cycle with all other members of the family Chlamydiaceae (1). Among chlamydiae affecting humans, Chlamydia trachomatis is the most clinically and epidemiologically relevant as a cause of oculogenital infections, including nongonococcal urethritis and cervicitis, lymphogranuloma venereum, and trachoma. Chlamydia pneumoniae is known to be involved in community-acquired pneumonia, pharyngitis, bronchitis, and sinusitis. In addition, the avian pathogen Chlamydia psittaci has well-documented zoonotic potential, causing human psittacosis (ornithosis), which may present as a generalized and life-threatening pneumonia (2).
C. abortus typically occurs in ruminants such as sheep and goats and is the leading cause of enzootic abortion of ewes (EAE) worldwide (3). Even in the absence of elevated abortion rates, the agent was shown to be widespread in German sheep flocks, with 50% of flocks testing PCR positive and 94% harboring seropositive animals (4). Afterbirths and fetuses of abortion cases can contain heavy loads of the pathogen and represent the major source of transmission to susceptible humans and naive ewes, as well as environmental contamination (3). Inhalation of infective aerosols by pregnant women poses the risk of severe infection, including spontaneous abortion, stillbirth, and septicemia (3, 5, 6). However, very little is known about the clinical relevance, epidemiology, and transmission of human C. abortus infection, since specific diagnostic tools are currently not available (3).
Recently, it was shown that relatively small doses of intranasally inoculated C. abortus organisms induced latent infection in nonpregnant ewes (7). When latently infected ewes became pregnant, this resulted in placental infection and consequent abortion, whereas animals infected with large doses were better protected and showed a much lower abortion rate. While laboratory diagnosis of EAE can be conducted with DNA- or protein-based tests, serology remains the preferred option in many laboratories (3). Despite limited sensitivity and specificity, the complement fixation test (CFT) is still the procedure most widely used to detect infection and determine vaccination titers (3, 8). A test based on polymorphic membrane protein 12G (Pmp12G) in an enzyme-linked immunosorbent assay format (3, 9) became commercially available in 2015. However, serological assays based on a panel of both surface and virulence-associated C. abortus antigens have not been established until now.
To extend the spectrum of potential diagnostic marker proteins, we identified 48 immunoreactive proteins by two-dimensional immunoblot analysis and screening of a C. abortus gene expression library in a previous project (10). From these, we have selected nine proteins for recombinant synthesis and further evaluation. These comprised (i) three surface proteins, specifically, the major outer membrane protein (MOMP), macrophage infectivity potentiator (MIP), and Pmp13G; (ii) three virulence-associated proteins, namely, the homologs of Chlamydia protease-like activity factor (CPAF), translocated actin-recruiting phosphoprotein (TARP), and secreted inner nuclear membrane-associated Chlamydia protein (SINC) (11), and (iii) three hypothetical proteins, specifically, CAB031, CAB821, and CAB408, two of which (CAB821 and CAB408) are predicted to be secreted by the type III secretion system (12). For a comprehensive analysis of the ovine and human antibody responses to these antigens, we used the so-called line immunoassay since this format allows the simultaneous detection of antibodies to multiple antigens in a single run (13). Characterized serum samples from (i) experimentally infected sheep, (ii) naturally infected sheep, (iii) infected humans, (iv) healthy blood donors and individuals with chlamydial infections other than C. abortus, and (v) humans at risk of exposure to C. abortus were analyzed.
MATERIALS AND METHODS
Serum samples.
The human and animal serum samples used in this study were taken from already existing serum collections of previous studies (4, 5, 7, 10, 13, 14). Human serum samples were anonymized, and their use was approved by the local ethics committee of the University of Ulm (96/09) and the local ethics committee of the University Hospital Jena (2525-04/09). Table 1 gives an overview of the serum samples used for this study and lists their main characteristics. Further details are described below.
TABLE 1
| Serum group | Reference(s) | ni (ns)b | Characteristic(s) |
|---|---|---|---|
| Animals | |||
| Experimentally infected sheep | 7 | 5 (35) | Inoculation with 5 × 103 IFU, abortion/stillbirth; histopathological findings in placenta specimen, significant amounts of C. abortus DNA in PCR |
| 5 (35) | Inoculation with 5 × 105 IFU, abortion/stillbirth; histopathological findings in placenta specimen, significant amounts of C. abortus DNA in PCR | ||
| 10 (70) | Inoculation with 5 × 107 IFU, normal lambing; no histopathological findings in placenta specimen, no significant amounts of C. abortus DNA in PCR | ||
| 3 (21) | Uninfected, asymptomatic sheep with normal lambing | ||
| Naturally infected sheep | 10 | 11 | Sheep from German flocks with endemic C. abortus infections; abortion, vaginal or rectal swabs positive for C. abortus by PCR |
| 4 | 29 | Sheep from German flocks with endemic C. abortus infections; normal lambing, vaginal or rectal swabs positive for C. abortus by PCR | |
| Negative and specificity control groups | 10 | 25 | Sheep from German flocks with endemic C. abortus infections; normal lambing, vaginal or rectal swabs negative for C. abortus by PCR |
| 4, 10 | 11 | Sheep from German flocks with endemic C. abortus infections; normal lambing, vaginal or rectal swabs negative for C. abortus and positive for C. pecorum by PCR | |
| 4 | 5 | Sheep from German flocks with endemic C. abortus infections; normal lambing, vaginal or rectal swabs negative for C. abortus and positive for C. psittaci by PCR | |
| Humans | |||
| Infected humans | 5 | 2 | Women with septic abortion, PCR positive for C. abortus |
| Humans at risk of exposure | 4, this study | 88 | Shepherds and veterinarians, unknown clinical history, close contact with sheep flocks with endemic C. abortus infections proven by PCR |
| Negative-control group | This study | 20 | Healthy blood donors, 10 female and 10 male |
| Specificity control group | 13 | 20 | Clinically symptomatic patients with infection due to C. pneumoniae proven by PCR |
| 13 | 20 | Clinically symptomatic patients with infection due to C. trachomatis proven by PCR | |
| 14, 31 | 3 | Clinically symptomatic patients with infection due to C. psittaci proven by PCR |
a
Animal and human serum samples are listed with their origins, numbers, and specific characteristics.
b
ni is the number of individuals, and ns the number of serum samples investigated; they are equal, except for the experimentally infected group of sheep, as serum samples were collected at seven different times following inoculation.
Animal serum samples. (i) Experimentally infected sheep.
Serum samples were obtained from three different groups of experimentally infected sheep as described in a previously published experimental model of latency (7). In that study, animals received different intranasally administered doses of infectious C. abortus elementary bodies (EBs) 8 weeks before mating and were monitored and bled over several months during pregnancy. Here, we investigated serum samples from 10 experimentally challenged sheep that underwent abortion, as well as 10 experimentally inoculated sheep that lambed normally with clinically healthy offspring. Three uninfected sheep served as a negative-control group. Serum samples were obtained at 0, 2, 5, 12, 21, 29, and 47 weeks after inoculation, with abortion and lambing occurring around 26 to 30 weeks postinoculation (p.i.) (Table 1).
(ii) Naturally infected sheep.
Serum samples were taken from 32 German sheep flocks with endemic C. abortus infections (4). Eleven serum samples originated from sheep with abortion and rectal or vaginal swabs positive for C. abortus by PCR. Twenty-nine serum samples originated from clinically asymptomatic sheep with vaginal and rectal swabs positive for C. abortus by PCR. Serum samples from asymptomatic, C. abortus PCR-negative sheep (n = 25), as well as serum samples from sheep positive for Chlamydia pecorum (n = 11) and/or C. psittaci (n = 5) served as negative and specificity controls (Table 1).
Human serum samples. (i) Pregnant women with septic abortion.
Serum samples were collected from women who had undergone septic abortion due to C. abortus as proven by PCR (5).
(ii) Shepherds and veterinarians.
We investigated 88 serum samples obtained from shepherds and veterinarians with close contact with infected sheep from flocks with endemic C. abortus infections and cases of abortion. The serum samples were collected during meetings of shepherds and veterinarians in parallel with the study on sheep by Lenzko et al. between 2009 and 2011 (4).
(iii) Negative and specificity control groups.
Serum samples were obtained from healthy blood donors (n = 20; 50:50 male-to-female ratio), as well as from clinically symptomatic patients with infections due to C. pneumoniae (n = 20), C. trachomatis (n = 20), or C. psittaci (n = 3), as proven by PCR (14).
Gene cloning and expression of recombinant antigens.
Nine immunoreactive C. abortus antigens were selected on the basis of previous work by Forsbach-Birk et al. (10) to be recombinantly expressed for the production of a line immunoassay (13). These were CAB048 (MOMP; Gene ID no. 3337752), CAB080 (MIP; Gene ID no. 3337460), CAB281 (Pmp13G; Gene ID no. 3337669), CAB712 (CPAF homolog; Gene ID no. 3337730), CAB167 (TARP homolog; Gene ID no. 3337791), and CAB063 (SINC homolog; Gene ID no. 3337689), as well as hypothetical proteins CAB031 (Gene ID no. 3337919), CAB821 (Gene ID no. 3338163), and CAB408 (Gene ID no. 3337407). All antigens except CAB031, which was truncated at its C terminus, were expressed as full-length proteins and highly purified by chromatographic methods (15).
Production of the C. abortus line assay.
The line assay enables the simultaneous detection of antibodies to selected antigens. All of the nine recombinant antigens (MOMP, MIP, Pmp13G, CPAF, TARP, SINC, CAB031, CAB821, and CAB408) were deposited in a line format on nitrocellulose membranes in separate lanes with a dispense platform. A reaction control; three conjugate controls for the detection of human immunoglobulin IgG, IgA, and IgM; and a cutoff line were also applied. For each C. abortus protein, individual dilutions and buffers were used. The membranes were saturated with a milk solution and cut into test strips.
Incubation of line immunoassays and readout of human and animal serum samples.
Strips were incubated with either human or animal serum samples (both diluted 1:100) overnight (human serum samples) or for 1 h (animal serum samples) at room temperature, allowing specific antibodies to bind to the C. abortus antigens. After a repetitive washing step with a phosphate buffer for 3 × 5 min, a peroxidase-labeled rabbit-anti-human IgG (diluted 1:100) or polyvalent rabbit anti-sheep antibody (DakoCytomation A/S, Glostrup, Denmark) was added (diluted 1:1,000) and the strips were incubated for 45 min at room temperature. After a second washing step, binding of specific antibodies was detected by the use of tetramethylbenzidine incubated for 8 min at room temperature. Strips were first examined for positive bands visible to the naked eye before being analyzed by intensity measurement. Intensities of reactive bands were measured with an OpticPro S28 scanner (Plustek, Norderstedt, Germany) and recomScan software (BioSciTec GmbH, Frankfurt, Germany) according to the manufacturers' instructions. Bands were considered positive if the ratio of the intensity of the antigen band to the intensity cutoff minus 15% tolerance was ≥1.0; this is referred to here as the cutoff-adjusted optical density (OD).
Statistical analysis.
Statistical analysis was performed with Microsoft Excel 2013. The Mann-Whitney U test was used to calculate the level of significance. Statistical significance was accepted as a P value of ≤0.05.
RESULTS
Animal serum samples. (i) Aborting experimentally infected sheep show strong and long-lasting antibody responses to surface and virulence-associated, as well as hypothetical, C. abortus proteins.
Experimental intranasal inoculation of small or moderate doses of infectious C. abortus EBs (5 × 103 and 5 × 105 inclusion-forming units [IFU], respectively) resulted in abortion in a majority of the animals. Their placental tissue was investigated by histopathology, immunohistochemistry, and quantitative real-time PCR, which revealed pathological tissue findings typical of EAE and evidence of C. abortus in placental tissue samples as described previously (7). Here, we analyzed serum samples from sheep with abortion following the inoculation of small (n = 5) and medium (n = 5) doses of EBs. A late antibody response not detectable until week 21 p.i. and peaking at the time of abortion at 29 weeks p.i. proved characteristic of these animals (Fig. 1A). An increase in antibodies to Pmp13G was detected in 50% of the sheep at 21 weeks p.i. (see Table S1A in the supplemental material), with bands being markedly more intense than those of the nonabortion group of experimentally infected sheep (P ≤ 0.02) (Fig. 2A). At the time of abortion at 29 weeks p.i., serum samples from all of the animals contained antibodies to Pmp13G, with OD values higher than those of the nonabortion group (P ≤ 0.02). In addition, strong and intense bands with distinctly higher OD values than those of the nonabortion group were found for the other surface antigens MOMP (P ≤ 0.02) and MIP (P ≤ 0.02). Apart from those, we were also able to measure higher OD values for the virulence-associated antigens CPAF (P ≤ 0.02), TARP (P ≤ 0.02), and SINC (P ≤ 0.02) (Fig. 1B), as well as for the hypothetical protein CAB031 (P ≤ 0.02) (Fig. 1C). Strong bands were still present after 47 weeks p.i., especially for the surface antigens MOMP (P ≤ 0.02) and Pmp13G (P ≤ 0.02), in a majority of the animals (Fig. 2A; see Table S1A in the supplemental material).
FIG 1
FIG 2
(ii) Normally lambing experimentally infected sheep show a rapid but short-term immune response to surface antigens.
Sheep experimentally inoculated with a large dose (5 × 107 IFU) of C. abortus EBs lambed normally, with healthy offspring and no evidence of placental pathology (7). These sheep (n = 10), which cleared the infection, showed a pattern of humoral immune response clearly different from that of animals that underwent abortion. Two weeks p.i. (6 weeks before mating), animals showed band patterns with increased OD values for the surface antigens MOMP (no statistically significant difference [n.s.]), MIP (n.s.), and Pmp13G (P ≤ 0.02), the latter being positive in 90% of the sheep (Fig. 2B; see Table S1B in the supplemental material). Band intensity rapidly decreased below the level of detection at subsequent sampled time points, with little or no detection of any response at the time of lambing. Uninfected animals kept under controlled laboratory conditions showed no measurable antibodies to the selected antigens (Fig. 2C) and are therefore not listed in Table S1 in the supplemental material.
(iii) Aborting naturally infected sheep show antibodies to surface and virulence-associated, as well as hypothetical, antigens.
Serum samples obtained from naturally infected ewes at the time of abortion were analyzed (n = 11). Compared to asymptomatic sheep that were PCR negative for C. abortus or PCR positive for C. pecorum or C. psittaci (n = 41 in total), we observed greater antibody reactivity to surface antigens MOMP (P ≤ 0.02), MIP (P ≤ 0.02), and Pmp13G (P ≤ 0.02); to virulence-associated antigens CPAF (P ≤ 0.02) and SINC (P ≤ 0.02); and to hypothetical protein CAB031 (P ≤ 0.02) (Fig. 2D and E; see Fig. S1 in the supplemental material). In total, 54.5% of the naturally infected sheep exhibited antibodies to MOMP, 72.3% exhibited antibodies to MIP and Pmp13G, 54.5% exhibited antibodies to CPAF, 72.3% exhibited antibodies to SINC, and 63.6% exhibited antibodies to CAB031 (data not shown).
(iv) Asymptomatic C. abortus PCR-negative and C. pecorum- or C. psittaci-positive sheep show little nonspecific antibody reactivity.
Serum samples from 41 asymptomatic sheep that were C. abortus PCR swab negative (n = 25) or C. pecorum (n = 11) or C. psittaci swab positive (n = 5) were investigated for the presence of antibodies to the antigen panel. We observed only single nonspecific antibody binding in individual sheep, but none of them showed more than one positive band. In total, 2.4% of the animals showed detectable antibody reactivity to MIP, Pmp13G, CPAF, or TARP, and none of the serum samples reacted with MOMP, SINC, or CAB031 (Fig. 2F).
(v) Asymptomatic carriers of C. abortus may show a weak-to-moderate antibody response.
Serum samples from 29 ewes that did not abort during the current lambing season but had positive C. abortus PCR results from vaginal or rectal swabs were tested. We observed that 10.3% of the animals showed antibody reactivity to MOMP, 27.6% showed reactivity to MIP, and 17.2% showed reactivity to Pmp13G. Humoral immune responses to CPAF, TARP, and CAB063 could be detected in 31.0, 27.6, and 13.8% of the sheep, respectively. Bands for CAB031 were present in 13.8% (data not shown).
Human serum samples. (i) Women who have aborted as a result of C. abortus infection reveal an antibody response to surface antigens of the bacterium.
Because of the rarity of the disease in humans, only two serum samples obtained from women who aborted as a result of C. abortus infection were available for analysis. Both samples revealed an immune response to the surface antigens MOMP, MIP, and Pmp13G. One of them showed antibody reactivity to the hypothetical protein CAB821, which is predicted to be type III secreted (Fig. 3A; see Fig. S2 in the supplemental material).
FIG 3
(ii) C. abortus Pmp13G is highly specific.
Blood donors failed to exhibit any measurable antibody response to any of the antigens included, whereas we observed sporadic cross-reactivity covering almost all of the antigens when testing serum samples from patients infected with chlamydiae other than C. abortus (Fig. 3B; see Fig. S2 in the supplemental material). Notably, surface antigen Pmp13G was the only non-cross-reacting antigen, which indicates its potential as a specific marker of C. abortus infection (see Table S2 in the supplemental material).
(iii) One shepherd showed a specific antibody response to C. abortus.
We analyzed serum samples obtained from shepherds and veterinarians (n = 88) who had close contact with sheep flocks with a high prevalence of C. abortus infection (4). Only a minority showed a detectable antibody response to any of the C. abortus antigens. In total, we detected a responses to MOMP in 6.9%, MIP in 14.9%, CPAF in 1.2%, TARP in 5.8%, and SINC in 4.6%. In addition, responses to hypothetical proteins CAB031 and CAB821 were found in only 3.5 and 1.2%, respectively (Fig. 3C; see Fig. S2 in the supplemental material). A single shepherd showed measurable amounts of IgG antibodies to multiple surface and virulence-associated proteins, including MOMP, MIP, Pmp13G, and SINC (Fig. 3C), which suggests that transmission had occurred.
DISCUSSION
To analyze the kinetics of antibody development that occurs during ovine abortion, we compared consecutive serum samples from latently infected aborting sheep with serum samples from sheep that had been inoculated with a large dose of C. abortus and lambed normally. We were able to show that, around the time of abortion, aborting sheep developed a strong antibody response to surface proteins MOMP, MIP, and Pmp13G, as well as to virulence-associated proteins CPAF and TARP and the SINC homolog CAB063. However, in the majority of experimentally infected aborting animals, antibody generation was not detectable until week 13 of gestation (week 21 p.i.). This is in agreement with the observations that chlamydial growth and pathology in the placenta are not evident any earlier than day 90 of gestation (7, 16) and that severe placentitis with massive chlamydial replication occurs in late pregnancy, when infected ewes are undergoing hormonal and immunological changes. As a consequence, detection of virulence-associated antibodies around the time of abortion may reflect the increased expression of virulence-associated proteins that are required to regulate and sustain intraplacental infection (17). Among these, TARP is a type III secreted protein that was shown to modulate host cell cytoskeleton function in C. trachomatis infection (18, 19). CPAF was initially described as a chlamydial protease degrading host cell transcription factors; however, its impact on virulence and pathogenicity is currently controversially discussed (20). SINC is a type III secreted protein targeting the nuclear membrane of infected cells, which may modulate the nuclear envelope function (10, 11). Here, we have demonstrated that virulence-associated antibodies are generated predominantly in animals that have aborted. This report therefore confirms the observation that virulence-associated proteins, including the newly described SINC homolog of C. abortus (CAB063), are immunogenic in ovine abortion (10). Although ewes that have aborted are considered protected from further abortion due to C. abortus, they represent a major reservoir of the pathogen for animals and humans and therefore need to be rapidly identified. As a major conclusion, detection of antibodies to both surface and virulence-associated proteins in a ewe may indicate imminent or recent abortion, since we have shown that levels of antibodies to virulence-associated proteins rapidly decreased below the level of detection within the subsequent observation period, while antibodies to surface antigens persisted. Further studies are needed to clarify whether antibodies to virulence-associated antigens in EAE may become a useful marker suggesting the implementation of control measures at the flock level (3) such as segregation of seropositive animals to limit dissemination to susceptible animals or treatment to prevent abortion.
The surface antigens investigated in this study have been shown to be immunoreactive in C. abortus infections (10), as well as in other chlamydial infections (13, 21, 22). MOMP makes up 50 to 60% of the total protein mass of the outer membrane of chlamydiae (23). The type V autotransporter Pmp13G (24) is a member of the Pmp family, representing proteins that play an important role in the pathogenesis of chlamydial infections. Several Pmps of C. trachomatis, C. pneumoniae, C. psittaci, and C. abortus have been shown to elicit a humoral immune response (13, 25), with PmpD of C. trachomatis being described as a pan-neutralizing antigen (26). Pmps are differentially expressed during the chlamydial developmental cycle (27, 28) and are therefore suspected to play a role in antigenic diversity and evasion of the host immune response. Pmp21 of C. pneumoniae has been described as an invasion protein that recruits the epidermal growth factor receptor for host cell entry (29).
In agreement with a study by Longbottom et al. (7), an early but transient antibody response to surface proteins was observed in sheep that lambed normally after being experimentally inoculated with large doses of C. abortus. The authors suggested that animals inoculated with large doses had, in principle, been vaccinated, since the dose was equivalent to that used in commercial live attenuated vaccines (3). Our data support this equivalence, as the absence of virulence factor-associated antibodies and the rapid decrease in antibody levels below the limit of detection suggest loss of the immunogenic stimulus and indicate elimination of the pathogen. The finding that 90% of normally lambing experimentally infected animals showed a rapid antibody response to Pmp13G (CAB281) could indicate a protective role of neutralizing antibodies to this antigen during clearance of the infection. In aborting experimentally infected animals, we have demonstrated that all of them developed antibodies to Pmp13G around the time of abortion and that these antibodies were still present in 90% of the animals 18 weeks after abortion. As sheep that have aborted will normally not abort again, we speculate that Pmp13G antibodies may contribute to the prevention of reinfection and abortion.
The antibody responses observed in experimentally infected animals correspond well to the present results obtained with serum samples from German sheep flocks with a high prevalence of C. abortus infection. More than 70% of the aborting ewes presented antibodies to Pmp13G and the virulence-associated SINC homolog at the time of abortion. As stated above, simultaneous detection of antibodies to surface and virulence-associated proteins may serve as a criterion to identify aborting animals and those on the verge of abortion. In contrast, nonaborting asymptomatic carriers of C. abortus either remained serologically negative or developed only a weak-to-moderate antibody response with much less prominent band patterns than aborting animals. Therefore, we can conclude that mere colonization with C. abortus cannot be reliably detected with serological tools. On the flock level, it is therefore not possible to identify asymptomatic C. abortus shedders serologically, even though recent abortion leads to significantly higher ODs of reactive antigen bands than mere colonization (see Fig. S1 in the supplemental material).
The CFT is the only test currently recognized by the World Organization for Animal Health for diagnosing ovine chlamydiosis (8). However, concerns about cross-reactivity preclude its use for species-specific diagnosis, as the antigen used includes a heat-resistant lipopolysaccharide that is present in all members of the family Chlamydiaceae (30). Further systematic studies of the sensitivity and specificity of the most promising antigens, such as Pmp13G, CPAF, and SINC or a combination of them, including quantification of band ODs, are needed to compare their diagnostic potential with that of CFT and recently developed serodiagnostic assays (3).
Concerning human C. abortus infection, pregnant women exposed to the pathogen run a substantial risk of developing severe infection and abortion, even though knowledge about the epidemiology of C. abortus infections in humans is poor. In most of the cases reported, a diagnosis was established through culture, PCR, or immunohistochemical analysis of placental tissue following abortion (5, 6). It is hard to deny that earlier microbiological diagnosis and earlier adequate antibiotic treatment would have substantially improved the clinical course of infection. Both of the patients examined here presented antibodies to MOMP, MIP, and Pmp13G. Even though two serum samples from confirmed infection would not justify definitive conclusions in terms of sensitivity, we suggest that the Pmp13G response is highly specific for C. abortus infection, since neither healthy blood donors nor patients suffering from other chlamydial infections revealed (cross-reactive) antibodies to Pmp13G in their serum samples. In contrast, the use of MOMP and MIP seems to be of limited value for species-specific serodiagnosis, as antibodies to these proteins were also detected in patients with infections with chlamydiae other than C. abortus.
Clinically relevant human C. abortus infections not associated with pregnancy have only sporadically been reported to date. We provide serological evidence that antigenic exposure to C. abortus may lead to a specific humoral immune response outside pregnancy. Nevertheless, the case of a male shepherd who presented antibodies to Pmp13G and the SINC homolog appears to be an exception, since he was the only one in a group of 88 individuals.
In summary, we have analyzed the humoral responses during both animal and human C. abortus infections. In animals that had aborted, we observed a strong antibody response to surface and virulence-associated proteins. Comparing experimentally infected animals with either asymptomatic infection or abortion, we were able to show that antibodies to virulence-associated proteins were raised predominantly in animals that aborted. The surface protein Pmp13G of C. abortus seems to be a sensitive and highly specific immunogen in animal infection. Even though the group of humans investigated here is too limited to make definitive conclusions on antigen sensitivities in humans, Pmp13G has proven to be a highly specific antigen that warrants further investigation. Further studies are needed to clarify whether antibodies to Pmp13G are protective and may contribute to the prevention of reinfection of sheep and whether they are suitable as a diagnostic marker in both sheep and humans.
ACKNOWLEDGMENTS
This study was supported by the German BMBF (Bundesministerium für Bildung und Forschung, Federal Ministry of Education and Research), project funding reference numbers 01KI1011C to A.E. and 01KI1001C to K.B., as well as The Scottish Government's Rural and Environment Science and Analytical Services Division (RESAS) to D.L.
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. E. Soutschek and J. Maile are employees of MIKROGEN Molekularbiologische Entwicklungs-GmbH, Neuried, Germany. We have no other conflicts of interest in relation to this work.
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Information & Contributors
Information
Published In
Journal of Clinical Microbiology
Volume 54 • Number 7 • July 2016
Pages: 1883 - 1890
Editor: E. Munson, Wheaton Franciscan Laboratory
PubMed: 27194684
Copyright
© 2016 American Society for Microbiology.
History
Received: 16 February 2016
Returned for modification: 8 March 2016
Accepted: 9 May 2016
Published online: 24 June 2016
Contributors
Editor
E. Munson
Editor
Wheaton Franciscan Laboratory
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Citations
Citation
2016.
Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay. J Clin Microbiol
54:.
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