Galápagos Penguin (Spheniscus mendiculus), Flightless Cormorant (Phalacrocorax harrisi), and Waved Albatross (Phoebastria irrorata) are among the most vulnerable species to natural and anthropogenic factors in the Galápagos Islands. In 2017, a dedicated study was conducted to detect Chlamydiaceae on cloacal swabs collected from 59 albatrosses, 68 penguins, and 10 cormorants in different islands and sites in the Galápagos Archipelago. A real-time PCR method targeting the conserved 23S ribosomal RNA gene of the Chlamydiaceae family detected the presence of the bacterium only in albatrosses from Punta Suárez, Española Island, with 21 positive samples (35.6%), whereas negative results were obtained with available real-time PCR systems specific to Chlamydia psittaci and Chlamydia abortus. Multilocus sequence typing (MLST) of the most strongly positive samples revealed a new sequence type closely related to the recently described avian strains of C. abortus. For a quick identification, a new real-time PCR system that allows the detection of all strains (avian and ruminant) belonging to the C. abortus species has been developed. Applied to a second set of samples from 31 albatrosses collected at Punta Suárez, Española Island, in 2018, the new real-time PCR system confirmed the presence of this bacteria in this group of birds, with the same new MLST sequence type.

The Galápagos Islands are located in the eastern Pacific Ocean, about 1,000 km west of the mainland coast of Ecuador. Animal species in this volcanic archipelago have high levels of endemism (Wiedenfeld and Jiménez-Uzcátegui 2008), including endemic marine birds (Harris 1973). These species are threatened by climate change, introduced new species, and human interaction, as well as multiple pathogens and parasites (Jiménez-Uzcátegui et al. 2019).

The obligate intracellular bacteria Chlamydiaceae are the etiologic agents of chlamydiosis in wild and domestic birds, mammals, and humans. The Chlamydiaceae family comprises the Chlamydia genus, which includes 14 recognized species and the very recently described Chlamydiifrater genus (Vorimore et al. 2021). Different Chlamydia species are hosted by birds, including among others, Chlamydia psittaci, a species long identified in a wide range of birds and with proven zoonotic potential, Chlamydia avium, Chlamydia gallinacea (Sachse et al. 2014, Aaziz et al. 2015), and more recently, Chlamydia abortus, which includes avian strains alongside the hitherto well-known ruminant strains (Zareba-Marchewka ̢ K et al. 2021), as recently detected from Polish wild waterfowl (Szymańska-Czerwińska et al. 2017). Avian strains of C. abortus can be distinguished from ruminant strains by distinct multilocus sequence typing (MLST) sequences and by the presence of a plasmid only detected in avian strains (Szymańska-Czerwińska et al. 2017).

To better characterize the chlamydial diversity in marine birds of the Galápagos Islands, we focused on three endemic bird species: Waved Albatross (Phoebastria irrorata), Galápagos Penguin (Spheniscus mendiculus), and Flightless Cormorant (Phalacrocorax harrisi). These three groups of birds had been studied in the past, including investigation for C. psittaci, the only chlamydia species known to be associated with birds at that time (Padilla et al. 2003; Travis et al. 2006a, b).

In 2017, cloacal swabs were collected from 59 Waved Albatrosses captured in Punta Suarez (Española Island); from 68 Galápagos Penguins captured in Caleta Iguana, Puerto Pajas (Isabela Island), and Marielas Islets; and from 10 Flightless Cormorants captured in Carlos Valle-Escondida (Fernandina Island) and Punta Albemarle (Isabela Island). The birds were captured and sampled as part of the Galápagos National Park Directorate and Charles Darwin Foundation research programs by specialized Galápagos National Park rangers according to the instruction of Charles Darwin Foundation scientists in the nesting areas. The swabs were taken by veterinarians experienced in handling wild birds. The birds were clinically evaluated during capture and none showed clinical signs. We extracted DNA with a commercial kit (QIAamp DNA Mini Kit, Qiagen, Courtabœuf, France) and analyzed it with a Chlamydiaceae real-time PCR as described by Ehricht et al. (2006). We detected Chlamydiaceae only in Waved Albatross, with 36% (21/59) of specimens positive (Table 1).

Table 1

Chlamydiaceae detected in samples from Waved Albatross (Phoebastria irrorata), Galápagos Penguin (Spheniscus mendiculus), and Flightless Cormorant (Phalacrocorax harrisi) sampled on the Galápagos Islands in 2017 and from Waved Albatross sampled in 2018 by 23S real-time PCR for Chlamydiaceae and our sucB real-time PCR for Chlamydia abortus (avian and bovine strains).

Chlamydiaceae detected in samples from Waved Albatross (Phoebastria irrorata), Galápagos Penguin (Spheniscus mendiculus), and Flightless Cormorant (Phalacrocorax harrisi) sampled on the Galápagos Islands in 2017 and from Waved Albatross sampled in 2018 by 23S real-time PCR for Chlamydiaceae and our sucB real-time PCR for Chlamydia abortus (avian and bovine strains).
Chlamydiaceae detected in samples from Waved Albatross (Phoebastria irrorata), Galápagos Penguin (Spheniscus mendiculus), and Flightless Cormorant (Phalacrocorax harrisi) sampled on the Galápagos Islands in 2017 and from Waved Albatross sampled in 2018 by 23S real-time PCR for Chlamydiaceae and our sucB real-time PCR for Chlamydia abortus (avian and bovine strains).

On analysis with previously published C. psittaci, C. avium, and C. abortus species-specific real-time PCR systems (Pantchev et al. 2009, Zocevic et al. 2013), all Waved Albatross samples tested negative. To clarify the species identity of the chlamydia strains hosted by these specimens, we partially sequenced the 16S ribosomal (r)RNA and 23S rRNA genes from the three positive DNA samples with the highest DNA load (17-4795_M045, 17-4795_M054, and 17-4795_M086). Amplification was performed as previously reported with primers 16S-rp2 and 16SF2-23SIgR (Aaziz et al. 2015). Sequencing of both DNA strands was performed at Eurofins Genomics (Ebersberg, Germany). All sequences (GenBank accession nos. MW995984–MW995986 and MW996893–MW996895) proved identical. A blastn search (National Center for Biotechnology Information 2022) revealed highest 16S rRNA sequence homology with C. psittaci and C. abortus species (>99%) and highest 23S rRNA sequence homology with C. abortus (98.4%; data not shown).

For better characterization, the MLST profiles of two of the strongest positive samples (17-4795_M045 and 17-4795_M048) were determined according to Pannekoek et al. (2010). This MLST genotyping method targets seven housekeeping genes—gatA, oppA, hflX, gidA, enoA, hemN, fumC—that were amplified and sequenced with primers and conditions described on the Chlamydiales MLST website (Jolley et al. 2018). Sequencing of both DNA strands was performed by Eurofins, and the numbers for alleles and the sequence type (ST) were assigned in accordance with the Chlamydiales MLST database. The MLST sequences obtained for the two samples were identical, with new sequences identified for each gene and with, in final, a new ST attributed (ST311; Table 2). We carried out comparative phylogenetic analysis of the concatenated MLST sequences of these two samples and a large panel of C. psittaci and C. abortus strains, including avian and ruminant strains. Briefly, DNA sequences were aligned by ClustalW available in MEGA7 (Kumar et al. 2016), and the best maximum likelihood model with the lowest Bayesian information criterion was applied. Trees were then inferred by the maximum likelihood method on the basis of the Tamura three-parameter model. This result showed the albatross specimens to be grouped alongside the avian strains of C. abortus detected in Polish wild waterfowl (Szymańska-Czerwińska et al. 2017; Fig. 1).

Table 2

Multilocus sequence type (ST) sequences for samples M45 collected from Waved Albatross (Phoebastria irrorata) sampled in the Galápagos Islands in 2017 (17-4795_M45, 17-4795_M48) and 2018 (20-3126_C79, 20-3126_D07).

Multilocus sequence type (ST) sequences for samples M45 collected from Waved Albatross (Phoebastria irrorata) sampled in the Galápagos Islands in 2017 (17-4795_M45, 17-4795_M48) and 2018 (20-3126_C79, 20-3126_D07).
Multilocus sequence type (ST) sequences for samples M45 collected from Waved Albatross (Phoebastria irrorata) sampled in the Galápagos Islands in 2017 (17-4795_M45, 17-4795_M48) and 2018 (20-3126_C79, 20-3126_D07).
Figure 1

Phylogenetic analyses of (A) concatenated sequences of seven multilocus sequence typing housekeeping gene fragments (enoA, fumC, gatA, gidA, hemN, hflX, and oppA) and (B) the plasmid sequences (707 bp) for four specimens (17-4795_M45, 17-4795_M48, 20-3126_C79, 20-3126_D07) from Waved Albatross (Phoebastria irrorata) sampled on the Galápagos Islands in 2017 and from representative sequences of Chlamydia psittaci and both avian and ruminant C. abortus strains. Trees with the highest log-likelihood are shown. The percentage of replicate trees in which the associated taxa clustered together is shown next to the branches (1,000 bootstraps; only values above 50% are shown). The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016). Albatross sequences are identified by an arrow, and avian strains of C. abortus are identified by a star.

Figure 1

Phylogenetic analyses of (A) concatenated sequences of seven multilocus sequence typing housekeeping gene fragments (enoA, fumC, gatA, gidA, hemN, hflX, and oppA) and (B) the plasmid sequences (707 bp) for four specimens (17-4795_M45, 17-4795_M48, 20-3126_C79, 20-3126_D07) from Waved Albatross (Phoebastria irrorata) sampled on the Galápagos Islands in 2017 and from representative sequences of Chlamydia psittaci and both avian and ruminant C. abortus strains. Trees with the highest log-likelihood are shown. The percentage of replicate trees in which the associated taxa clustered together is shown next to the branches (1,000 bootstraps; only values above 50% are shown). The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016). Albatross sequences are identified by an arrow, and avian strains of C. abortus are identified by a star.

Close modal

Previous studies focusing on the search for C. psittaci had been conducted between 2001 and 2004 on penguin, cormorant, and albatross populations in the Galápagos (Padilla et al. 2003, Travis et al. 2006a, b). The methods applied, including serologic analysis or direct detection by PCR on cloacal, pharyngeal, and choanal swabs, produced different results from ours. Positive serologic results, as will as the detection of specific C. psittaci sequences, were previously obtained from penguins and cormorants, whereas in our study, albeit with PCR only and not serology, failed to find evidence of Chlamydiaceae in these two bird populations. However, for albatross, our study detected Chlamydiaceae, whereas negative results had been obtained previously. Our study was carried out more than 15 yr after the previous studies. It is possible that 1) the agents circulating at the earlier time are no longer present, 2) our sample set was too small to detect the circulation of the bacteria in penguins or cormorants, or 3) tools used in previous studies (serology in particular) were not specific for Chlamydiaceae or detected other Chlamydiales families not investigated in our study.

Currently available real-time PCR systems are inadequate to detect newly identified avian strains of C. abortus (Pantchev et al. 2009, 2010); therefore, we developed a new real-time PCR system allowing the simultaneous detection of ruminant and avian strains belonging to this species. In brief, after the alignment of whole genome sequences of C. abortus and C. psittaci, we developed a new real-time PCR system that uses the dihydrolipoamide acetyltransferase (sucB) gene with a primers and probe combination for the specific detection of all strains belonging to the C. abortus species. Primers Cabortus_sucB_F (5′-TTG CTG TGG GAA CAG AAC GT-3′) and Cabortus_sucB_R (5′-CTC GCT AAA TCC GCA AGC TTC-3′) and a specific probe Cabortus_sucB_P (5′-[FAM] TGT TCC AGT GAT TCG TGA GGC CGA [BHQ1]-3′) were designed by the Primer3-Plus (Untergasser et al. 2007) software. Real-time PCR amplifications were performed as described by Aaziz et al. (2022). The specificity of the new sucB real-time PCR system, detecting multiple C. abortus strains but not strains of other Chlamydia spp., is presented in Supplementary Material Table S1. The sensitivity and detection limit are indicated in Supplementary Material Figure S1 and Table S2. This new real-time PCR system allowed the correct detection of C. abortus species in albatrosses sampled in 2017 and from albatrosses sampled (n=31) from Punta Suárez, Española Island, Galápagos, in 2018. With the sucB real-time PCR, three of four albatrosses that tested positive for Chlamydiaceae with 23S real-time PCR were confirmed as carrying C. abortus (Table 1), confirming the circulation of this chlamydial species in the albatross population in Galápagos.

The MLST sequences of two positive samples from the catches conducted in 2018 (samples 20-3126_C79 and 20-3126_D07) confirmed circulation of ST311 in this population. An in-house primer set that allows detection by conventional PCR of the plasmid harbored by avian C. psittaci and C. abortus strains was used as described in Aaziz et al. (2022) to analyze the samples genotyped by MLST (17-4795_M45, 17-4795_M48, 20-3126_C79, and 20-3126_D07). A single new sequence was detected in these samples collected in 2017 and 2018 (GenBank accession nos. MZ073730–MZ073733). The plasmid sequence is closely related to sequences detected in Polish C. abortus samples (Fig. 1), with 94.4% and 98.3% identity to the plasmid sequences associated with C. abortus 15-49d3 and 15-70d24 strains, respectively.

Data on prevalence and host and geographic distributions of the recently characterized avian strains of C. abortus are limited. These strains have so far been detected in wild birds (Anatidae and Corvidae) from Poland, Sweden, or both (Blomqvist et al. 2012; Szymańska-Czerwińska et al. 2017) and in captive parrots (Longbottom et al. 2021). Those studies concerned only cases diagnosed in Europe. Our study adds samples from the Pacific Ocean, suggesting an active circulation of these avian strains of C. abortus among birds in different geographic areas. Bird migration might explain the circulation of the strains: the Galápagos Albatross breeds in the equatorial islands of the Galápagos and feeds on the South American coasts, where exchanges with other bird communities are possible. We found a single and unique MLST sequence type from two catches conducted in 2017 and 2018. It would be interesting to sample albatross specimens from different colonies and different geographic areas to see whether they harbor similar chlamydia strains. Previously, assignment of samples to avian C. abortus has not been possible because of the lack of specific tools. The new real-time PCR system developed in this study allowed us to detect and characterize such strains rapidly.

Unfortunately, no strains could be recovered from the second set of samples for which an additional swab in conservative buffer was taken, probably because of a low concentration of the bacteria, the delay in storing the samples before import, or both. It would be beneficial to analyze such samples thoroughly. To date, the whole genome sequences of the Polish strains (Zar̢ eba-Marchewka et al. 2019, 2021) and the recently reassigned C. abortus 84-2334 strain (Longbottom et al. 2021) are available, alongside their MLST sequences. In the study by Szymańska-Czerwińska (2017), three groups of avian strains were highlighted inside the C. abortus species: 1V and G1, closer to the ruminant strains and including the recently reassigned C. abortus 84-2334 strain, and G2, corresponding to the strains mainly hosted by Anatidae so far. From our MLST data, the strains hosted by the Galápagos Albatross are related to group G1, but on a distinct branch (Fig. 1).

It is worth remembering that none of the birds showed any clinical signs of disease at the time of capture. Likely, the presence of these chlamydia are associated with an asymptomatic carriage, as has been described for seabirds and wild waterfowl from which related strains have been isolated. Any effect of the C. abortus strains in the wild albatrosses cannot yet be evaluated. A zoonotic effect could neither be excluded nor confirmed. Precautions should be taken during the capture and sampling of these birds.

We thank our donors, the Galápagos Conservation Trust, the Truell Charitable Foundation, the Leona M. and Harry B. Helmsley Charitable Trust, Lindblad–National Geographic, the Penguin Fund of Japan, and the Peregrine Fund. We also thank Seishi Sakamoto for his financial support of the Marine Birds Project. We are grateful to our collaborators and more than 90 assistants and volunteers who helped in the research from 2009 to 2021. We thank the Ministerio del Ambiente for granting research authorizations and permissions (MAE-DNB-CM-2016-0043) and the Galápagos National Park Directorate (PC-25-20, PC-34-19, PC-0518, PC-10-17, PC-51-16, PC-35-15). We thank the Charles Darwin Research Station for authorizations and permissions. This publication is contribution number 2402 of the Charles Darwin Foundation for the Galápagos Islands. None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-21-00163.

© Wildlife Disease Association 2023

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Author notes

6These authors contributed equally to this work;

Supplementary data