Co-Occurrence of Chlamydia suis DNA and Chlamydia suis- Specific Antibodies in the Human Eye

Abstract

Chlamydia suis is a swine pathogen that causes economic losses due to reproductive failure. Recently, C. suis has been detected in human eyes. However, knowledge of the zoonotic potential is still limited. C. suis infections in swine could present a risk for public health because (1) tetracycline-resistant C. suis strains are emerging in the pork industry, (2) tetracycline resistance gene transfers in vitro from C. suis to the human pathogen Chlamydia trachomatis and as previously demonstrated, (3) C. suis and C. trachomatis can be both present in the human eye. Pig farmers were sampled during a seminar in West-Flanders. Conjunctival swabs for detection of C. suis and C. trachomatis and for the detection of mucosal antibodies against C. suis and C. trachomatis were collected. The farmers completed a questionnaire designed to assess information on the following: (1) the health status of their pigs, (2) administration of veterinary drugs, (3) their professional and nonprofessional activities, (4) general health status, (5) smoking habits, (6) use of medication, (7) allergies, and (8) clinical signs/history. Thirty-three on 40 (82.5%) farmers participated. None of the conjunctival swabs contained C. trachomatis DNA and mucosal antibodies against C. trachomatis were not detected. Six of 33 (18.2%) farmers had C. suis DNA in their eyes and 22 of 33 (67%) swabs contained C. suis-specific mucosal antibodies. The older the farmer, higher the chance of finding C. suis antibodies in the eye. There was a significant correlation between the presence of conjunctivitis in the pigs and the occurrence of C. suis DNA in the eye of their owner. This study shows that C. suis may transfer from pigs to the human eye as specific mucosal antibodies were detected in conjunctivae of pig farmers. Veterinarians, general practitioners, and occupational physicians should be aware of the zoonotic potential of C. suis.

Introduction

C hlamydia suis is an obligate intracellular Gram-negative bacterium. The pig is the only known natural host of C. suis. C. suis in pigs has been associated with asymptomatic infections but also with a variety of clinical signs such as conjunctivitis, rhinitis, pneumonia, enteritis, reproductive disorders such as irregular return to estrus, early embryonic death in inseminated sows and inferior semen quality in boars (decrease of sperm cell motility and dead of sperm cells) (Rogers et al. 1993, 1996, Rogers and Andersen 1996, 1999, 2000, Szeredi et al. 1996, Di Francesco et al. 2008, Schautteet et al. 2010, 2013, Englund et al. 2012, Rypula et al. 2014, Hoffmann et al. 2015, Chahota et al. 2018). C. suis is phylogenetically highly related to the human pathogen Chlamydia trachomatis (Joseph et al. 2016) revealing 83–94% genome identity depending on the C. trachomatis strain. C. trachomatis serovars A-C infect the conjunctivae leading to infectious blindness (trachoma). Serovars D-K and L1-3 cause urogenital infections in men and women. C. suis is not classified into serovars, but as mentioned it also causes eye infections and reproductive failure although in sows and boars (Schautteet and Vanrompay 2011, Chahota et al. 2018).

C. suis infections could be successfully treated with tetracyclines until the appearance of a tetracycline-resistant (Tc^R) phenotype, which was first isolated on pig farms in Iowa and Nebraska (Andersen and Rogers 1998). Soon thereafter, Tc^R C. suis strains appeared in other countries including Belgium, Cyprus, Germany, Israel, Italy, Switzerland, and The Netherlands (Di Francesco et al. 2008, Borel et al. 2012, Schautteet et al. 2013, Donati et al. 2016, Wanninger et al. 2016). Resistance was acquired via horizontal gene transfer of the class C gene [tet(C)]-containing cassette.

The emergence of Tc^R C. suis strains raises considerable concern. In vitro studies have already shown that the [tet(C)]-containing cassette can be transferred between C. suis and C. trachomatis (Suchland et al. 2009). Recently, Dean et al. (2013), found C. suis messenger RNA (mRNA) in the eyes of Nepalese trachoma patients. C. suis DNA and/or viable organisms were also recovered from the eyes of two Belgian slaughterhouse employees (De Puysseleyr et al. 2014a) and from the eyes of seven Belgian pig farmers (De Puysseleyr et al. 2015). As far as we know, Tc^R C. suis phenotypes have not yet been found in humans. However, contact between Tc^R and tetracycline-sensitive Chlamydia spp. in different settings, including farms, veterinary clinics, and slaughterhouses, may lead to transfer of the resistance gene and associated phenotype, which could then be selected for in patients treated with tetracycline. In this case, the treatment of Chlamydia infections in patients would be hampered, which could result in more severe complications.

Recent studies showed that C. suis infections are highly prevalent in the Belgian pork industry (De Puysseleyr et al. 2014a, 2015) and C. suis has been found in the eyes of humans occupied in the pork industry. However, C. suis could have ended up in the eyes by touching the face with contaminated hands. Indeed, C. suis was present on 9 of 14 tested contact surfaces and hand to face (eye) contamination thus might have occurred (De Puysseleyr et al. 2014a). So, strictly taken, we do not know for sure whether C. suis causes an infection of the human eye. Isolation of C. suis from field samples is fastidious and challenging (De Puysseleyr et al. 2016). Therefore, we investigated the occurrence of C. suis eye infections in Belgian pig farmers by demonstrating both C. suis DNA and C. suis-specific mucosal antibodies using two in-house developed diagnostic tests. At the same time, we examined the co-occurrence of C. trachomatis infections in the human eye.

Materials and Methods

Samples

Pig farmers were sampled (informed consent) in Rumbeke, West-Flanders, Belgium during a seminar organized by the Flemish government on ventilation and climate regulation in pig barns. Conjunctival swabs for Chlamydia real-time PCR were collected by rotating a sterile aluminum shafted rayon tipped swab (Copan) on the conjunctiva of the left eye lid of the volunteer. The procedure was repeated with a new swab for the right eye. Swabs were stored in 2 mL DNA/RNA stabilization reagent (Roche, Mannheim, Germany). The procedure was repeated to obtain mucosal samples for antibody detection by enzyme-linked immunosorbent assay (ELISA). Swabs for antibody detection were submerged in 2 mL cOmplete^™ Protease Inhibitor Cocktail (Roche), prepared by dissolving one protease inhibitor tablet in 10 mL sterile phosphate buffered saline (PBS, pH 7.4; Sigma-Aldrich, Overijse, Belgium). Samples were transported on ice and upon arrival in the laboratory, left and right conjunctival swabs of each individual were pooled and subsequently stored at −80°C. Farmers filled in a veterinary and medical questionnaire, designed to assess information on the general health status, use of medication and clinical signs/history of their pig herd and to assess information on their professional (work environment) and nonprofessional activities, general health status, smoking habits, use of medication, allergies, and clinical signs/history. The study was approved by the human Ethical Committee of Ghent University (EC2014/1205).

Chlamydia detection in eye samples

DNA extraction was performed using the G-spin Total DNA Extraction Mini Kit (Goffin Molecular Biotechnologies, Beek, The Netherlands) according to the instructions of the manufacturer. Samples were tested using a 23S ribosomal RNA (rRNA) based C. suis-specific real-time PCR (De Puysseleyr et al., 2014b). Briefly, real-time PCR was performed with the Rotor-Gene Q Instrument (Qiagen Benelux, Venlo, The Netherlands) using 25 μL of a reaction mixture containing 2 μL of DNA template, 4 μL of primer mixture (300 nM forward and reverse primer), 2.5 μL of the C. suis 23S-probe A and B (200 nM) to detect all C. suis strains, 12.5 μL of absolute qPCR mix (Thermo Scientific, Acros Organics, Geel, Belgium), and 1.5 μLDNAse and RNAse-free water. The cycling conditions were as follows: 95°C for 15 min, 50 cycles of 95°C for 15 s, 60°C for 60 s. All default program settings were used. Standard graphs of the cycle threshold (Ct) values, obtained by testing tenfold serial dilutions (10⁸–10¹) of the purified species-specific inhibition control plasmid, were used for quantification. DNA was always tested in the presence of control plasmid (50 copies/μL) to check for PCR inhibitors. Samples were also tested using the C. trachomatis-specific CE-In Vitro Diagnostic (CE-IVD) certified Presto real-time PCR Kit (Goffin Molecular Diagnostics, Houten, The Netherlands), which detects the C. trachomatis cryptic plasmid. The PCR contains an inhibition control for evaluating the presence of polymerase inhibitors. The PCR was performed according to the manufacturer's instructions. Samples with a Ct-value below 35 cycles, were retested twice. Only repeatedly positive samples were judged as positive. Genomic DNA of the C. suis reference strain S45 (ATCC VR-1474) was used as positive control DNA (10⁵ infection forming units per reaction) and DNAse-RNAse-free water was used as negative control.

Detection of C. suis and C. trachomatis-specific antibodies in mucosal swabs

The presence of antibodies against C. trachomatis was examined by a commercial C. trachomatis-specific ELISA (C. trachomatis-IgG-ELISA plus; Medac Diagnostics, Wedel, Germany) that detects antibodies against a C. trachomatis-specific peptide of the major outer membrane protein (MOMP). The presence of antibodies against C. suis was examined by an in-house developed C. suis-specific ELISA (polymorphic membrane protein C [PmpC] CS IgG ELISA) (De Puysseleyr et al. 2018) based on the use of an immunodominant C. suis-specific B cell epitope (SQQSSIAS) of the PmpC. The B cell epitope was synthetically designed (Pepscan Presto, Lelystad, The Netherlands), preparative high performance liquid chromatography purified (purity up to 99%) and analyzed by mass spectometer-ultra performance liquid chromatography. The peptide contained an N-terminal acetyl group and was C-terminal attached to polyethylene pins via incorporation of an extra cysteine. Hundred μg of the peptide was coupled to each pin. The peptide-coated pins were assembled on a 96-well polyethylene carrier (pin peptide ELISA format) for ELISA.

For the PmpC CS antibody ELISA, pins were first blocked for 3 h at room temperature at 100 rpm using 250 μL per well of PBS (pH 7.2; Invitrogen, Carlsbad, CA) supplemented with 5% bovine serum albumin (BSA; Sigma-Aldrich) and 0.1% Tween 20 (Sigma-Aldrich). Afterward, the pins were washed three times for 10 min at 100 rpm with PBS containing 0.1% Tween 20 (washing buffer). After washing, pins were incubated overnight (4°C at 100 rpm) with 150 μL mucosal swab content diluted 1:50 in PBS supplemented with 3% BSA and 0.1% Tween 20 (dilution fluid). After washing, pins were incubated (1 h at room temperature at 100 rpm) with 150 μL/well of horseradish peroxidase labeled goat anti-human-IgG (H+L) that detects IgG, IgM, and IgA (Invitrogen), diluted 1/500 in dilution fluid. After washing, pins were incubated (room temperature at 100 rpm) with 150 μL/well of the substrate H₂O₂-chromogen ABTS mixture (KPL, Gaithersburg, MD) where after the optical density (OD₄₀₅) could be determined (Tecan GENios Plus, Mechelen, Belgium). The cutoff value was the mean absorbance of negative controls ± twice the standard deviation (SD). Values above the cutoff value were regarded as positive. Diluted protease inhibitor (1/50 in PBS) served as negative control. Positive controls, being mucosal fluid or sera from C. suis-infected humans are currently unavailable.

Detection of antibodies against the Chlamydia MOMP in mucosal swabs

Nobody ever investigated the presence of C. suis-specific antibodies in the human eye and positive controls for the PmpC CS antibody ELISA were thus unavailable. Therefore, the following strategy was used to verify negative PmpC CS antibody ELISA results. Mucosal swabs that were negative in the PmpC CS antibody ELISA were subsequently analyzed by an ELISA detecting IgG (H+L) antibodies against another Chlamydia target antigen, namely the MOMP of C. suis reference strain S45. The MOMP of Chlamydia contains genus-, species-, and family-specific epitopes. Diluted (1/50) protease inhibitor served as negative control and human sera from patients infected with C. trachomatis (kindly obtained from S.M.) were used as positive control. Patients had reacted positive (at a serum dilution of 1/100) in the commercial C. trachomatis-specific ELISA (C. trachomatis-IgG-ELISA plus; Medac Diagnostics).

For the MOMP ELISA, first recombinant MOMP (rMOMP) was produced in pcDNA4::MOMPS45 transfected COS-7 cells as described previously (Vanrompay et al. 1998). The MOMP ELISA was performed as described by Schautteet et al. (2011). Briefly, 96-well plates were coated with rMOMP of C. suis reference strain S45. Plates were blocked overnight (4°C) with PBS supplemented with 5% BSA. The next day, wells were washed with PBS and samples diluted 1/50 in PBS +3% BSA +0.05% Tween20 (dilution buffer) were added and allowed to incubate (1 h at 37°C). After washing, goat anti-human IgG (H+L) antibody (1/1000 in dilution fluid; Invitrogen) was added for 1 h at 37°C. After washing, ABTS substrate solution (Lucron Bioproducts, Sint-Martens-Latem, Belgium) was added and the OD₄₀₅ was measured (Tecan GENios Plus). The cutoff value was the mean absorbance of negative controls ± twice the SD. Values above the cutoff value were regarded as positive.

Statistical analysis

The outcome variables were single or mixed infection, Chlamydiaceae species found in the eye, PCR and/or ELISA positive and the read outs from the veterinary and medical questionnaire. Associations between discrete variables were analyzed by Pearson chi-square or Fischer's exact test using SPSS statistics 24. A p value of <0.05 was used as the cutoff for determining statistical significance.

Results

Chlamydia detection

Thirty-three of 40 (82.5%) farmers participated in the study and none of the eye swabs was found positive using the C. trachomatis real-time PCR. However, the C. suis-specific real-time PCR could identify 6 out of 33 (18.2%) positive samples with Ct-values ranging between 31 and 34.

Chlamydia antibody detection

C. trachomatis antibodies were not detected in mucosal swabs. Twenty-two of 33 (67%) samples reacted positive in the C. suis-specific ELISA (PmpC CS IgG ELISA). The OD₄₀₅ of the positive samples ranged between 0.205 and 1.171. Five of six (83%) C. suis PCR positives were also positive in the PmpC CS antibody ELISA and the OD₄₀₅ values of these samples ranged between 0.251 and 0.520. Eleven of 33 (33%) samples were determined to be negative in the C. suis-specific ELISA, which was also confirmed by the rMOMP ELISA.

Questionnaire

Ten of 33 (30%) farmers had a low animal contact frequency (<30 h/week). Only one of them (10%) indicated to have eye problems (dry, irritated, itchy, tired, and/or sore eyes) in the last 2 weeks. This person was PCR negative but reacted positive (OD₄₀₅ 0.315) in the PmpC CS antibody ELISA. Nine of these 10 (90%) farmers indicated to have no eye problems. One of these nine persons (11.1%) was PCR positive (Ct value of 32.75) but reacted negative in the PmpC CS antibody ELISA and three of these nine persons (33.3%) were PCR negative but reacted positive (OD₄₀₅ 0.205, 0.311 and 0.278) in the PmpC CS antibody ELISA.

Twenty-three of 33 (69.7%) farmers had a high animal contact frequency (>30 h/week). Nine of them (40%) reported to have eye problems (dry, irritated, itchy, tired, and/or sore eyes) in the last 2 weeks. Five of those nine (55.5%) persons were both PCR and ELISA positive. Four of those nine (44.4%) farmers were PCR negative but all reacted positive in the PmpC CS antibody ELISA. Fourteen of 23 (60%) farmers had no eye problems (dry, irritated, itchy, tired, and/or sore eyes) in the last 2 weeks. All of them were PCR negative and nine (64.2%) reacted positive in the PmpC CS antibody ELISA.

We found a significant correlation [t(28_z) = −2.30; p < 0.05; m⁺ = 49.0; SD⁺ = 9.42; m⁻ = 36.3; SD⁻ = 10.42] between the age of the pig farmer and the result of the PmpC CS antibody ELISA. The older the farmer (>35 years of age), the higher the chance of finding C. suis antibodies in the eye. In addition, a significant correlation was found between the presence of C. suis DNA in the human eye and conjunctivitis in the pigs [χ²(1) = 4.90; p = 0.09]. Moreover, If C. suis DNA was present in the human eye, 100% of their animals showed conjunctivitis. However, if C. suis DNA was absent in the human eye, conjunctivitis was also noticed in the pigs, but only in 10–39% of their animals. We found no significant correlation between animal contact frequency and eye-related clinical signs in the farmer. There was no correlation between a positive test (ELISA or PCR) and animal contact frequency. Furthermore, no correlation was found between other clinical conditions (for instance respiratory distress) in humans and animal contact frequency. There was no correlation between the age of the farmers and their physical health status.

Discussion

All eye swabs were negative in the C. trachomatis real-time PCR, which is perhaps not surprising as nowadays, C. trachomatis eye infections (trachoma) especially occur in developing countries (recently reviewed by Mohammadpour et al., 2016) and as far as we know, the last report on C. trachomatis eye infections in Belgium dates back to 1994, when C. trachomatis was found in 29% of 104 adult patients with chronic conjunctival irritation without obvious diagnosis (Van Ginderdeuren and Missotten 1994). However, diagnosis was performed by May-Grunwald-Giemsa and fluorescence staining (monoclonal antibody against the chlamydial lipopolysaccharide) of conjunctival scrapings. These tests are not C. trachomatis-specific and can even detect C. suis.

During our study, results from the C. suis-specific real-time PCR revealed 6 out of 33 (18.2%) positives, but since the values ranged between 31 and 34, this might point more toward a past or chronic infection rather than an active eye infection. Of the 27 PCR negative swabs, only 2 had a Ct-value of 35 (35.1 and 35.85). Thus, C. suis nucleic acids did appear in the human eye, as also observed in other recent studies (Dean et al. 2013, De Puysseleyr et al. 2014a, 2015). However, we were still not sure if we were dealing with a real eye infection. Eye-related clinical complaints were mentioned in the medical questionnaire but a clinical examination was not performed in this study. In future studies, involving a larger human risk population, as well as randomly selected individuals, the expertise of an ophthalmologists would be helpful to implement a clinical grading system, analogue to the WHO simplified grading system, which is used for the examination of ocular C. trachomatis infections (Thylefors et al. 1987).

A significant correlation was found between the presence of C. suis DNA in the human eye and conjunctivitis in the pigs. However, pigs were not examined in this study as <20% of the farmers allowed us to sample their animals. We realize that other infectious or even noninfectious reasons can cause conjunctivitis in swine. Thus, we cannot really draw conclusions on the correlation between conjunctivitis in pigs and the presence of C. suis DNA in the human eye.

In future studies, culture could be included. However, in the past, only small amounts of viable C. suis have been found in the eyes of persons working in the pork industry (De Puysseleyr et al., 2014a, 2015). The latter is not unexpected because (1) C. suis is difficult to grow in cell culture (Rogers et al. 1993, Sandoz and Rockey 2010) and (2) C. suis growth efficiency can differ between strains, dependent on the cell line used (De Puysseleyr et al. 2016). The latter probably reflects the high genetic diversity within this species as demonstrated recently by Joseph et al. (2016), who performed a comparative genomic analysis of C. suis, by whole genome alignment of 12 C. suis strains.

The Chlamydia antibody detection showed one person being positive by PCR and negative by the PmpC CS antibody ELISA. This could reflect a very recent infection or perhaps a subclinical infection, as according to the medical questionnaire eye-related clinical complaints were absent in this person.

The samples that reacted negative in the C. suis-specific ELISA were further examined by the rMOMP ELISA and they were all negative in this test, confirming the C. suis-specific ELISA results and actually also confirming the results of the C. trachomatis-specific Medac ELISA. The rMOMP ELISA was used to verify PmpC CS antibody ELISA negative results in the absence of a positive control for the latter test. However, now that we have C. suis positive human control sera, the rMOMP ELISA would no longer be needed for a next study on a larger human risk population, which is important as 71% of the farmers indicated daily contact with other animals, as chickens, dairy cattle, small ruminants, dogs, and cats, all known to be hosts for Chlamydia spp. different from C. suis, which, however, also could be transmitted to humans (Hartley et al. 2001, Longbottom and Coulter 2003, Lagae et al. 2014, Rodolakis and Laroucau 2015).

Although the results of the questionnaire are promising and some significant correlations could be found, a subsequent larger population-based study could give more information. A larger study population might allow us to identify significant correlations between animal contact frequency, eye-related clinical signs, other clinical complaints, the farmer's age, and a positive test (ELISA or PCR). An important aspect that needs to be studied in a subsequent study, is the relationship between active C. suis eye disease (grading system) and ocular C. suis infection, as it is well known that there is a variable relationship between the signs of active trachoma and the detection of an ocular C. trachomatis infection at the individual level (Ramadhani et al. 2016). The same could be the case for C. suis eye infections as this species is phylogenetically highly related to C. trachomatis.

Conclusions

C. suis may transfer from pigs to the human eye as C. suis-specific mucosal antibodies were detected in this study from conjunctiva of pig farmers. Veterinarians, general practitioners, occupational physicians, and ophthalmologists should be aware of the zoonotic potential of C. suis. Collaborative research (One Health) is needed to study the clinical impact of C. suis eye infections in humans and to prevent the zoonotic transfer as it might present a risk for public health, certainly when Tc^R C. suis strains are involved.

Footnotes

Acknowledgments

The study was funded by the Federal Public Service of Health, Safety of the Food Chain and Environment (convention RF-10/6234) and the Flemish Fund for Scientific Research (FWO grant G.0776.13N).

Author Disclosure Statement

No competing financial interests exist.

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