Ovine Enzootic Abortion (OEA): a comparison of antibody responses in vaccinated and naturally-infected swiss sheep over a two year period

Background Prevention and control of ovine enzootic abortion (OEA) can be achieved by application of a live vaccine. In this study, five sheep flocks with different vaccination and infection status were serologically tested using a competitive enzyme-linked immunosorbent assay (cELISA) specific for Chlamydophila (Cp.) abortus over a two-year time period. Results Sheep in Flock A with recent OEA history had high antibody values after vaccination similar to Flock C with natural Cp. abortus infections. In contrast, OEA serology negative sheep (Flock E) showed individual animal-specific immunoreactions after vaccination. Antibody levels of vaccinated ewes in Flock B ranged from negative to positive two and three years after vaccination, respectively. Positive antibody values in the negative control Flock D (without OEA or vaccination) are probably due to asymptomatic intestinal infections with Cp. abortus. Excretion of the attenuated strain of Cp. abortus used in the live vaccine through the eye was not observed in vaccinated animals of Flock E. Conclusion The findings of our study indicate that, using serology, no distinction can be made between vaccinated and naturally infected sheep. As a result, confirmation of a negative OEA status in vaccinated animals by serology cannot be determined.


Background
Chlamydophila abortus (formerly Chlamydia psittaci serotype 1) is the most common infectious bacteria causing abortion in small ruminants in Switzerland with a previ-ous study demonstrating that 39% of the examined abortions in sheep and 23% in goats were caused by this agent [1]. In the Swiss canton of Graubünden, a mountainous region in the countries' east, the economic losses associ-ated with ovine enzootic abortion (OEA) are significantly higher than in other cantons [2].
Cp. abortus is generally introduced into immunologically naïve flocks by a latently infected animal with the agent being subsequently transmitted from aborting ewes via shedding of large amounts of infectious Chlamydia in the foetal membranes and in vaginal discharges [3]. In newly infected flocks, up to 30% of ewes may abort in the last trimester of gestation or give birth to weak or dead lambs. After abortion, ewes in these flocks may develop a protective immunity. Subsequent yearly losses in endemically infected flocks may decrease to a lower level (eg. 5-10%) with sheep either born into the flock or newly introduced animals likely to suffer abortions during their initial pregnancies [4,5].
Prevention and control of OEA is achieved by vaccination and/or treatment with oxytetracyclines [6]. Two vaccines against chlamydial abortion are licensed in Switzerland by the Federal Veterinary Office (FVO) in Berne. The first of these available was an egg-grown, formalin-inactivated, whole-organism vaccine (Ovax Clamidia, Fatro, Italy) which reduces the incidence of abortion in vaccinated herds but not completely [7][8][9][10]. Since December 2002, an avirulent, temperature-sensitive, live chlamydia vaccine (Ovilis ® Enzovax, Intervet, The Netherlands), which is marketed to induce strong long-lasting protection, has been made commercially available in Switzerland. The attenuated strain 1B, which forms the basis of this vaccine, was obtained from the virulent Cp. abortus strain AB7 by nitrosoguanidine mutagenesis [11][12][13].
In 2005, a small pilot study was undertaken to determine if administration of vaccines to protect sheep flocks from OEA would result in antibody levels in the complementfixation test (CFT) and in the competitive enzyme-linked immunosorbent assay (cELISA) tests similar to those following natural infection [14]. After vaccination with the inactivated vaccine (Ovax Clamidia) only one sheep developed a detectable antibody response. In contrast, vaccination with the attenuated live vaccine (Ovilis ® Enzovax) resulted in detectable antibody titers in all tested sheep.
The aim of this study is to investigate a larger number of sheep over a two-year period in the field to compare flocklevel ELISA responses between (a) vaccinated (live vaccine), (b) naturally infected and (c) non-infected sheep flocks. It was anticipated that the follow up study of the humoral responses could possibly discriminate between vaccinated and naturally OEA-infected sheep. An additional objective of the study was to attempt to detect chlamydiae and/or the attenuated strain of Cp. abortus used in the live vaccine in conjunctival swabs of sheep.

Results
Serological results and abortion cases cELISA classifications (frequency and proportion positive), median titer and respective range of positive classified sheep in flocks A, B, C, D and E over the four different investigation dates are shown in Table 1. The comparison between vaccinated and non-vaccinated animals in Flock B and E is shown in Table 2. Figure 1 shows the titer ranges (box plots) of all examined sheep in the five flocks over the four investigation dates.

Symbol legends
Box plot with 25th, 50th and 75th percentiles, and whiskers representing ~ 95% of the range

A1v
Moderate and severe outliers

Statistical comparison of mean titers
In flocks A (all animals vaccinated), C and D (no animals in both flocks vaccinated, Figure 1), differences in titer values between the sampling periods were always highly significant in the RM ANOVA model (p < 0.01). In Flock B, with a vaccination date between sampling periods 2 and 3, both vaccination status and an interaction term between vaccination and visit were statistically significant (p < 0.05). In Flock E, in which vaccination took place before the first sampling, both main effects were significant (time: p < 0.05; vaccination status: p < 0.01), while the interaction term was not.

PCR results of eye swabs
In

Discussion
This study represents the first longterm chlamydial serological study comparing vaccinated and non-vaccinated flocks in Switzerland. The investigations were undertaken in the canton Graubünden, where numerous chlamydial abortion cases in sheep were previously reported [1] and the highest seroprevalence (43%) for Cp. abortus in Swiss cantons was observed [2].
The results obtained from this study confirm the previous observations of the pilot study [14] that serology (cELISA) cannot be used to distinguish between sheep vaccinated with the live attenuated vaccine and naturally-infected sheep. The antibody value range in the recently vaccinated Flock A was comparable to Flock C in which acute infections of Cp. abortus occured at the same time. In Flock A, very high antibody levels (around 90%) were visible in every vaccinated sheep (n = 15), whereas antibody levels of sheep in the previous pilot study were somewhat lower (around 60%) 21 days post vaccination [14]. As chlamydial abortion was reported in Flock A in the past, sheep could have been already serologically positive before vaccination and the very high antibody levels could represent an overlay of both abortion and vaccine-associated antibody values. The mean antibody value of positive animals decreased in both flocks (A and C) from spring 2005 to spring 2006. A chlamydial abortion was diagnosed in one goat from Flock C in autumn 2006 explaining the increasing seroprevalence and antibody value in this group of animals at that time. The antibody values in the goats of Flock C after an acute infection with Cp. abortus were higher and persisted at a very high level (80 to 90%) over the observation period compared to the situation in sheep. No correlation with protection was seen however, as a chlamydial abortion occurred in a seropositive goat which had previously aborted. This observation was also made in other goat flocks in canton Graubünden (R. Thoma, personal communication). Goats treated with the live vaccine also aborted. In general, it is known that if Chlamydiae are introduced in a naive flock, the losses are much higher in goats (60%) than in sheep (30%). The differences between goats and sheep are consistent with previous records and to date remain unexplained [15,16].
Antibody levels of vaccinated ewes of Flock B ranged from negative to positive two and three years after vaccination, respectively. Questionable antibody levels are either attributed to undiagnosed Cp. pecorum infections [17] or are possibly due to the vaccination in spring 2003. In a similar situation to the naturally infected sheep (Flock C), a slow decrease of antibody values was observed over the sampling period. This observation strongly suggests that serology (cELISA) cannot be used to distinguish between sheep vaccinated with the live attenuated vaccine and naturally-infected sheep as anticipated in the previous pilot study [14]. As a direct consequence to this, the confirmation of negative OEA status in vaccinated animals by serology cannot be made. This is unfortunate as reliable confirmation is important if an abatement of OEA through assembly of OEA-free flocks is to be performed as undertaken by the Sheep and Goat Health Schemes in England and Wales and the Premium Health Scheme in Scotland.
Positive antibody values have been observed in the negative control flock (Flock D), which had not been vaccinated and was free from chlamydial abortion. An explanation for the observations of an increasing antibody value amongst this flock is that the animals may have asymptomatic intestinal infections with Cp. abortus as presumed in previous studies [17,19]. An alternative scenario is that the ewes were infected with a less virulent strain of Cp. abortus, which provokes seroconversion but no abortion [17,20]. Fluctuations in the antibody levels could be the result of bacterial shedding during oestrus which provokes an induction of antibody levels without causing abortion [21,22]. Unfortunately, little is still known at this time about the ability of Cp. abortus to persist in animals (and the anatomical location of this persistent infection) compared to other chlamydial species, which require more investigations.
In Flock E, the serological reaction of 13 selected vaccinated sheep and the 50 non-vaccinated sheep in the flock was evaluated. Surprisingly and in contrast to the observations in the previous pilot study [14] and in the two vac- Sampling of conjunctival swabs from sheep in Flock E was performed to detect and compare the presence of chlamydial DNA before and after vaccination. Furthermore, a possible excretion of the vaccine through the eye could be screened with this approach. Although chlamydiae were frequently detected by PCR in conjunctival swabs of sheep, the attenuated strain of Cp. abortus used in the live vaccine was not detected in swabs collected from vaccinated sheep. The incidence of Cp. abortus and Cp. pecorum and even C. suis in clinically healthy non-vaccinated sheep was previously observed in a recent study [23]. The significance of this possible new mode of transmission for OEA needs further investigation.

Conclusion
The findings in our study strongly suggest that serology (cELISA) cannot be used to distinguish between sheep vaccinated with the live attenuated vaccine and naturallyinfected sheep. The course of antibody levels, nevertheless, can vary between individual animals and flocks. Compared to sheep, goats displayed higher antibody levels, which persist over a longer time period but do not correlate with protection. The attenuated strain of Cp. abortus used in the live vaccine was not detected in eye swabs collected from vaccinated sheep.

Flock details
Five different sheep flocks in the canton Graubünden were followed over a two-year period with four flock visits. These five flocks were available for the study in spring 2005 through an established collaboration with veterinary authorities in the canton Graubunden. cELISA Serum samples were tested by the competitive enzymelinked immunosorbent assay (cELISA) using the monoclonal antibody mAb 188 directed against the variable segments 1 (VS1) and 2 (VS2) of the major outer mem-brane protein (MOMP) of Cp. abortus, according to the protocol of Salti-Montesanto et al. [17]. The results of the cELISA were expressed as 'percentage of inhibition' corresponding to the antibody concentration in the sample. Inhibition values above 55 per cent were considered positive for infection with Cp. abortus (positive cut-off) whereas inhibition values between 30 -55 per cent were classified as questionable, attributable to either Cp. abortus or Cp. pecorum, a widely distributed chlamydial agent in small ruminants causing diseases such as arthritis/conjunctivitis and pneumonia syndrome in lambs and also subclinical intestinal infections [18,19]. Inhibition values below 30 per cent were assumed to be negative [17,24].

PCR of eye swabs
Conjunctival swabs (Cytobrushes, Berdat Charles, Bourroux, Switzerland) were collected from Flock E before and after vaccination to investigate possible excretion of chlamydiae and/or the Cp. abortus vaccine strain through the eye. Before application of the vaccine, conjunctival swabs from every sheep in the flock (n = 118) were collected in autumn 2005. Five months following vaccination (spring 2006), the second conjunctival swab samples were taken from every sheep in the flock (n = 118). Cytobrushes were each placed in a 1.5-ml Eppendorf tube and stored at -80°C until further processing. DNA extraction from all swabs was performed as described previously [25] using a commercial DNA extraction kit (DNeasy Tissue Kit ® , Qiagen, Hombrechtikon, Switzerland).
The conjunctival swabs were investigated for the presence of chlamydial DNA by a Chlamydiales-order specific PCR targeting the intergenic spacer region (IGS) between chlamydial 16S and the 23S rRNA genes [26] and using primers cIGS1f (5'-CAA GGT GAG GCT GAT GAC-3') and cIGS2r (5'-TCG CCT KTC AAT GCC AAG-3'). PCR conditions are described elsewhere [26]. The identity of all positively tested IGS PCR products was determined by direct sequencing of the PCR product from both strands. Sequencing was performed with an ABI Prism 377 DNA sequencer (Applied Biosystems) or Applied Biosystems 3100 (Synergene Biotech). The obtained sequences were compared with the sequences available in GenBank using the BLAST server from the National Center for Biotechnology Information [27].

Investigation of abortion cases
Abortion cases in the flocks were further investigated for the presence of chlamydiae by routine bacteriology and immunohistochemistry of the placenta and the fetal organs (lung, liver, kidney) as described elsewhere [28].

Statistical analysis
Ewe ELISA antibody values were initially categorized into positive, questionable or negative as described previously  [17,24]. For the analysis, questionable and negative results were both interpreted as negative. Whole flock response patterns over time were visualized using box plots. For those sheep that were tested all four times, the proportion of positive ewes at each time point was compared within each flock using a Fishers Exact Test with exact p-values. In addition, the mean titers of those sheep were compared using a repeated measures ANOVA with animal ID, time (within animal repetition factor), vaccination status (flocks B and E only), and the interaction between time and vaccination (again only for flocks B and E).
Data were stored and handled in MS Excel, and analysed using the statistical software packages NCSS 2004 [29] and SPSS 14 [30]. The overall level of statistical significance was set to 0.05.