New insight on chlamydiae

Chlamydiae (in the Chlamydiales order, Chlamydiaceae family, Chlamydia and Chlamydiifrater genera) are a group of obligate intracellular bacteria distinguished by a biphasic developmental cycle comprising extracellular and intracellular stages. Chlamydiaceae representatives have relatively small genomes of about 1 Mbp, implying the absence of essential cellular pathways and consequential reliance on host cells for nutrients including amino acids, nucleotides and lipids. In the course of evolution, chlamydial genomes have undergone vast condensation, which was proven to derive from genome streamlining rather than degradation (39).

Chlamydiae are reported in various hosts living in different environments, including livestock and companion animals as well as wild species (3, 22). Chlamydial species with their main hosts are presented in Table 1. It is commonly known that most chlamydial taxons are highly capable of host switching. The advent of molecular techniques has enabled the detection of novel chlamydial species in a variety of host species; therefore, the host barriers for known Chlamydiaceae members may be less impenetrable than previously thought. Avian and mammalian subgroups should now be included (54) in an expanded version of the species definition of C. abortus given by Everett et al. (8), because a monitoring survey of wild birds including isolation and whole-genome sequencing (WGS) of avian C. abortus strains indicated this wider host range. Shedders of chlamydiae are widespread among wild animals, although the bacteria mostly inhabit those animals as commensal species without any negative impact on the host or sympatric species (44). However, Chlamydiaceae cause a number of important diseases both in animals and humans. Chlamydiosis can cause economic losses in livestock including ruminants (e.g. cattle or sheep) and poultry. Chlamydia gallinacea, as an emerging chlamydial agent, impacts poultry production parameters negatively, was also detected in cattle in China, and poses a risk to human health, which attracts the interest of researchers (10, 11, 12, 20, 28, 42, 43). Chlamydial infection is a potential risk to public health in wildlife hosts in addition to being one in farmed animals; the ongoing global process of habitat destruction as well as environmental and climate changes demand that urgent surveillance of chlamydial infection in these hosts be undertaken to better understand this risk.

Animals that are often latently infected or shed chlamydiae intermittently over a longer period of time are particularly dangerous to human health. The zoonotic potential of some chlamydial species is well documented in the literature (e.g. C. psittaci, C. abortus or C. pneumoniae), while for novel species the potential is still unclear or not fully confirmed (6, 29). The occurrence of chlamydiosis in humans depends on multiple factors including the intensity of exposure, microbial factors and route of transmission (34). Human infections are underdiagnosed and many are unreported because there is a lack of specific serological tools for detection of novel species and there are limited appropriate specimens available for real-time PCR testing.

The purpose of this article is to review the most important novel developments in the field of animal chlamydiae research.

Access to molecular methods and whole-genome sequencing greatly increases the amount of data on record about chlamydiae. Bioinformatics tools order this data and facilitate direct comparisons and evaluations of evolutionary relationships between different taxons and genotypes. As a result, the taxonomy of the Chlamydiaceae family has been constantly evolving. Since 1999 Chlamydiaceae has undergone a number of taxonomic reclassifications at the genus and species levels. The nomenclature has been under constant change in recent years. The genus Chlamydia was split into two genera, Chlamydia and Chlamydophila, based on analysis of the 16S and 23S rRNA genes (8). This change had been controversial for many years, until finally the chlamydial research community decided to restore the single genus, Chlamydia. Until recently, the Chlamydiaceae family was composed of this single genus, but it has now been extended to include a novel one (26, 36). Vorimore et al. (50) proved a new genus’ affiliation based on ultrastructural analysis, comparison of 16S and 23S rRNA gene sequences, and whole-genome analysis of new strains isolated from flamingos. The researchers made the case for Chlamydiifrater gen. nov. belonging to the Chlamydiaceae family besides the Chlamydia genus proposed two new species, Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov. New taxons were detected in the gastrointestinal tracts of flamingos in good health without clinical signs, and therefore they are considered commensal bacteria (50).

There are currently nine well-known species (C. trachomatis, C. suis, C. muridarum, C. pneumoniae, C. abortus, C. caviae, C. felis, C. pecorum and C. psittaci) and six new ones have been discovered in recent years. These new species have been recorded mainly in birds (C. avium, C. gallinacea and C. buteonis) and in snakes (C. serpentis and C. poikilotherma (previously named C. poikilothermis), one having been discovered in crocodiles (C. crocodili) (5, 19, 38, 40). Moreover, there are four new taxons with Candidatus status: Cand. C. corallus, and Cand. C. sanzinia were described in snakes, Cand. C. testudinis was detected in tortoises, and Cand. C. ibidis was reported in avian hosts (17, 25, 46, 51).

Discovering new chlamydial agents makes avian chlamydiosis (AC) much more complex than has been believed for decades. It transpires that not only C. psittaci, may be involved in the aetiology of AC in birds, overturning what was previously thought. Besides new avian taxons (C. avium, C. gallinacea, C. buteonis and Cand. C. ibidis), other well-known species primarily harboured by non-avian hosts (such as C. abortus, C. pecorum, C. trachomatis, C. suis and C. muridarum), might also be involved in AC (20). Recent research suggests that wild birds are common hosts for not only C. psittaci but also new avian taxons (e.g. C. gallinacea, C. avium or C. buteonis) (19, 38). Unfortunately, the impact of infection with these species on host health and condition remains unknown.

Recent reports on C. gallinacea shed new light on chlamydiosis in poultry and led to rejection of the hypothesis of the domination of a well-known species – C. psittaci – in poultry flocks. It has been proved that C. gallinacea is an emerging agent widespread in domestic and industrial poultry. Its predominant shedding in poultry was reported in Argentina, China, the Netherlands, Poland, the USA, Australia, Italy and Mexico (10, 13, 21, 28, 42, 45). Interestingly, it was also found in wild birds: the crimson rosella (Platycercus elegans) and galah (Eolophus roseicapillus) parrot species common in Australia (41, 42) and the woodcock (Scolopax rusticola) in South Korea (15). However, knowledge of the epidemiology of C. gallinacea in Psittaciformes is still limited. C. gallinacea is predominantly found in asymptomatic chickens but substantially lower body weight gains in experimentally infected SPF broilers and low mortality in embryonated eggs after yolk sac inoculation with C. gallinacea were noticed (10, 12, 43).

A phylogenetic intermediate position between C. psittaci and C. abortus is observed in another new taxon – C. buteonis – harboured by raptor birds and first isolated from a red-shouldered hawk (Buteo lineatus) in North America. Chlamydia buteonis was recovered from tissue of the carrier’s conjunctiva and content of the cloaca. This bacterium can cause conjunctivitis and/or respiratory signs or be a contributing factor to these clinical manifestations (19).

Chlamydia psittaci, a well-known causative agent of avian chlamydiosis and human psittacosis, is a genetically heterogeneous species. Sachse et al. (37) recently provided novel insights into the genetic diversity within this species. Based on analysis of whole-genome sequences of C. psittaci strains, the existence of four major clades (1–4) within this species was revealed. The authors reported that genomic divergence is connected with past host change and covers deletions in the plasticity zone, 3D structural variations in immunogenic domains of the outer membrane protein ompA, and various protein repertoires belonging to the Pmp and Inc families considered to include virulence factors. Chlamydia psittaci strains of clade 1 have limited genetic divergence and have been isolated both from avian (90% of all isolates being psittacine strains) and non-avian hosts, in the latter case as strains which were previously acquired from birds. This clade includes typical C. psittaci strains which are more virulent than strains of other clades. Clade 2 comprises only one psittacine strain, Mat116, and three strains of non-psittacine origin. Members of clade 3 present the most genetic divergence and have been isolated both from mammalian (such as sheep, cattle and human) and non-psittacine avian (duck strain) just as clade 4 does (having mainly pigeon strains). Both clades are differentiated by the loss of membrane attack complex/Perforin (MACPF) in the plasticity zone, as well as aberrant repertoires of Incs and Pmps (37).

Chlamydia abortus has only been detected in mammals, and animals in this class can also be hosts to C. psittaci, although the latter bacterium was considered until recently to be the most common species recorded in avian hosts. In contrast to C. psittaci, the genome of C. abortus is considered to be relatively stable with very little diversity and to undergo little or no recombination. In recent years a number of C. psittaci strains that differ from the classical avian C. psittaci have been identified in various countries. Based on comparative sequence analysis and multi-locus sequence typing, it was suggested that these atypical C. psittaci strains are more closely related to C. abortus species (30, 48). Research on wild birds in Poland revealed the existence of avian isolates being C. psittaci/C. abortus intermediates with worldwide distribution in various wild bird families (Anatidae, Corvidae and Rallidae) (44). Genomic analysis performed by Zaręba-Marchewka et al. (54) in agreement with analyses conducted by Longbottom et al. (22) confirmed that strains demonstrating features characteristic of both C. psittaci and C. abortus are recent evolutionary ancestors of C. abortus. The effect of these findings was that avian isolates previously referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates were classified as a new group called avian C. abortus. Whole-genome sequencing and comparative genomic analyses indicate this new subgroup of C. abortus to comprise strains previously classified as belonging to genotype F of C. psittaci, including Prk/Daruma and 84/2334 strains (9, 49), as well as strains representing genotypes G1, G2 and 1V of avian C. abortus (23, 54). The taxonomic definition of C. abortus species became outdated in the light of this and was amended to include two subgroups, mammalian and avian (2). Avian C. abortus strains can be found in birds, mainly in waterfowl (genotypes G1 and G2), corvids (genotype 1V) and parrots (Prk/Daruma and 84/2334 strains) and can be isolated from the cloaca and oropharynx (44).

Avian C. abortus strains, in contrast to classical C. abortus isolates of mammal origin, carry a plasmid the structure of which is most similar to that of C. psittaci plasmids (23, 54). Further studies on avian C. abortus plasmids could influence the development of new vaccines against avian C. abortus and/or C. psittaci strains, as protein plasmids are being used in this field (31, 54). Contrary to the case of C. psittaci, there is no evidence of a relationship between shedding of avian C. abortus and clinical signs in avian hosts. A key region of chlamydial genomes associated with pathogenesis is the plasticity zone (PZ) (1, 27, 35). The size of the PZ ranges from 6 to 83 kilobase pairs depending on the chlamydial species (55). In most chlamydial species, the boundaries of this region are acetyl-CoA carboxylase (accB) and einosine-5′-monophosphate dehydrogenase (guaB) genes. However, avian C. abortus representatives as well as other Chlamydia species hosted by birds (C. gallinacea, C. avium and C. buteonis) have a lost or truncated guaAB-add operon (1, 27). The plasticity zone includes several virulence factors including cytotoxin genes, adherence factor, and the MACPF and phospholipase D enzymes. Considering the size, avian C. abortus strains have a rather reduced PZ in comparison to most Chlamydia spp., but a more extensive zone when compared to the classical C. abortus S26/3 with its structure most similar to C. buteonis RSHA and C. psittaci 6BC. Interestingly, avian C. abortus strains carry the cytotoxin gene which is absent in classical C. abortus S26/3. However, in contrast to C. psittaci 6BC and C. buteonis RSHA, the MACPF gene is absent from the avian C. abortus PZ, as it is from C. abortus S26/3 (54).

Beside birds, poikilothermic animals are also an ecological niche for Chlamydiaceae. Shedders of Chlamydiaceae have been observed among reptiles including tortoises belonging to the Testudinidae family. Chlamydia pneumoniae is presently considered the dominant species in reptiles and is reported in snakes and chameleons. Interestingly, the existence of a new Candidatus Chlamydia species was recorded in tortoises in Italy, Poland and Germany (14, 25): a new chlamydial taxon closely related to C. pecorum. It could be widespread in tortoises showing clinical signs or free of signs; therefore, further investigations into its prevalence are needed. Three new taxons closely related to C. pneumoniae (Cand. C. corallus, Cand. C. sanzinia and C. serpentis) and a new species related to C. caviae (C. poikilotherma) were recently recognised in snakes (40, 46). Another new species, C. crocodili, hosted by Siamese crocodiles (Crocodylus siamensis) is closely related to C. poikilotherma and C. caviae, and its prevalence in other crocodiles species cannot be excluded. Infections in crocodiles could be asymptomatic or lead to kyphoscoliosis in juveniles, conjunctivitis, pharyngitis, ascites, depression, anorexia and death (5). The zoonotic potential of these new reptilian species has not yet been evaluated.

Zoonotic transmission of a few chlamydial species (C. psittaci, C. abortus and C. suis) is well-proven, while for the others the evidence is limited (e.g. C. felis and C. caviae). Evidence is also lacking on the zoonotic potential of novel reptilian and avian species (excluding C. gallinacea) (Table 1). Regarding this species, its zoonotic potential was suspected for the first time after the occurrence of atypical pneumonia in poultry slaughterhouse workers in France; however, this potential has not been definitively confirmed in the years since that occurrence, mainly because no species-specific serological method has been developed for detection of the immune response in human samples (18). Recently, C. gallinacea was detected by Marchino et al. (24) in sputum samples from farm workers having contact with infected poultry. For the first time, at international level, the hypothesis of the transmission of C. gallinacea from birds to humans was partially confirmed as phenomenon not restricted to France. Contrasting results were published by Dutch scientists, who failed to detect C. gallinacea in throat swabs taken from humans exposed to C. gallinacea- excreting poultry (13). It should be highlighted that there is no relation between the detection of C. gallinacea DNA in sputum and the appearance of clinical signs in humans. Therefore, in order to confirm the possibility of zoonotic transmission, further research on and isolation of C. gallinacea strains directly from human specimens is needed.

Chlamydia psittaci is a well-documented zoonotic avian pathogen, but avian shedders might be a source of transmission also to other animal species. Transmission of C. psittaci from birds to horses has been documented. The most pathogenic C. psittaci, 6BC, can be transmitted from equines to humans, as reported in Australia as the cause of an outbreak of respiratory illness in veterinary school students who had been exposed to equine foetal membranes (3). The mechanism of transmission between horses and humans is still not clear (34).

Chlamydia pneumoniae, a common chlamydial agent in humans, is also detected in animals (e.g. koalas); however, there are no reports of zoonotic cases. Genetic analyses of human C. pneumonia isolates confirmed that they are likely to have evolved from animal strains. Therefore animal isolates might be zoonotic, but it also cannot be ruled out that humans might be able to infect animals. Taking into account the common prevalence of this bacterium in people, zoonotic transmission would be difficult to confirm. Transmission between animals and humans has not been documented for C. muridarum or C. pecorum.