Aspects of the epidemiology of Chlamydia psittaci infection in a population of koalas (Phascolarctos cinereus) in southeastern Queensland, Australia

A population of free-ranging koalas in southeastern Queensland was examined to determine the prevalence of Chlamydia psittaci infections. Although C. psittaci was isolated from 46 of 65 (71%) koalas studied, only six (9%) of these had clinical signs of disease. Most adult females (82%) had back or pouch young present even though 67% of them were infected. There were no significant correlations between age, sex or site of sampling (urogenital versus conjunctival tissues) and the isolation of C. psittaci. No other important bacterial or fungal pathogens were isolated. The complement fixation test had a sensitivity of 7% and a specificity of 94% in detecting chlamydial infections, suggesting that it is unsuitable for use as a screening test. Chlamydia psittaci infection within this population appeared to represent a generally well-balanced host-parasite relationship and few animals had clinical signs of disease. Only four of 27 (15%) healthy koalas infected with C. psittaci followed for 24 wk after sampling developed eye disease or "dirty tail." Two koalas with keratoconjunctivitis recovered without treatment during the study period. Additional factors, including the stresses imposed by loss of habitat, may act to produce overt disease in koalas with latent C. psittaci infections.     

Ophthalmologic examination of the normal eye of the koala.

The ophthalmic examination of the koala (Phascolarctos cinereus) was undertaken to describe the normal in vivo structure. Twenty-two of 28 koalas examined were found to have normal eyes. Slit-lamp examination revealed unusual lamellae in the cornea, and mean keratometric readings were 59.3 +/- 3 diopters, corneal endothelial cell area of 381.4 microns2 +/- with a standard deviation of 46.8 microns2. No particular features were seen which would explain the susceptibility of this animal to ocular infection by Chlamydia psittaci.     

Evaluation of an enzyme immunoassay test for the diagnosis of Chlamydia psittaci infection in free-ranging koalas (Phascolarctos cinereus) in southeastern Queensland, Australia

The IDEIA Chlamydia Test, a commercially available antigen-capture enzyme-linked immunosorbent assay (ELISA) test, based on a monoclonal antibody for the detection of chlamydia in clinical specimens, was evaluated in a population of 65 free-ranging koalas in southeastern Queensland determined to be infected with Chlamydia psittaci. Compared to isolation of the organism in tissue culture, the sensitivity of the IDEIA test ranged from 3 to 11%, and the specificity from 90 to 97%. The results indicated that the IDEIA test is unsuitable for use as a diagnostic screening test for C. psittaci in free-ranging koalas.     

A Prototype Recombinant-Protein Based Chlamydia pecorum Vaccine Results in Reduced Chlamydial Burden and Less Clinical Disease in Free-Ranging Koalas (Phascolarctos cinereus)

Diseases associated with Chlamydia pecorum infection are a major cause of decline in koala populations in Australia. While koalas in care can generally be treated, a vaccine is considered the only option to effectively reduce the threat of infection and disease at the population level. In the current study, we vaccinated 30 free-ranging koalas with a prototype Chlamydia pecorum vaccine consisting of a recombinant chlamydial MOMP adjuvanted with an immune stimulating complex. An additional cohort of 30 animals did not receive any vaccine and acted as comparison controls. Animals accepted into this study were either uninfected (Chlamydia PCR negative) at time of initial vaccination, or infected (C. pecorum positive) at either urogenital (UGT) and/or ocular sites (Oc), but with no clinical signs of chlamydial disease. All koalas were vaccinated / sampled and then re-released into their natural habitat before re-capturing and re-sampling at 6 and 12 months. All vaccinated koalas produced a strong immune response to the vaccine, as indicated by high titres of specific plasma antibodies. The incidence of new infections in vaccinated koalas over the 12-month period post-vaccination was slightly less than koalas in the control group, however, this was not statistically significant. Importantly though, the vaccine was able to significantly reduce the infectious load in animals that were Chlamydia positive at the time of vaccination. This effect was evident at both the Oc and UGT sites and was stronger at 6 months than at 12 months post-vaccination. Finally, the vaccine was also able to reduce the number of animals that progressed to disease during the 12-month period. While the sample sizes were small (statistically speaking), results were nonetheless striking. This study highlights the potential for successful development of a Chlamydia vaccine for koalas in a wild setting.     

Some aspects of the immune response of koalas (Phascolarctos cinereus) and in vitro neutralization of chlamydia psittaci (koala strains)

Abstract Western-blot analysis was used to study the reaction of koala antisera, two specific polyclonal antibodies and one monoclonal antibody, with chlamydial antigens in koalas infected with Chlamydia psittaci . The koala sera recognized four C. psittaci surface antigens, corresponding to the major outer membrane protein (39.5 kDa), 31 kDa protein, 18 kDa protein and lipopolysaccharide. The S25-23 LPS specific monoclonal antibody inhibited chlamydial infection (55–67%) with both koala strains (type I and type II). Both koala antiserum and rabbit polyclonal antibodies against either type of chlamydia significantly reduced the number of infected cells resulting from type II infections at a dilution of 1 in 20. Rabbit antiserum against type II was effective in neutralizing infection by type II elementary bodies, but was less effective against type I infection. In addition, no koala antiserum was effective in neutralizing type I infection.     

Characterization of the koala biovar of Chlamydia pneumoniae at four gene loci--ompAVD4, ompB, 16S rRNA, groESL spacer region

Koalas are infected with two species of Chlamydia, C. pecorum and C. pneumoniae. While it is known that significant genetic diversity occurs in the C. pecorum strains infecting koalas, very little is known about the C. pneumoniae strains that infect this host. In the current study, 10 isolates of koala C. pneumoniae were analysed at four gene loci and found to be different to both the human and horse C. pneumoniae strains at all loci (biovar differences ranging from 0.3% at groESL up to 9.0% at ompAVD4). All koala biovar isolates studied were found to be 100% identical at ompAVD4 (all 10 isolates) and at ompB (all three isolates) gene. This lack of allelic polymorphisms at ompAVD4 has now been observed for koala C. pneumoniae, human C. pneumoniae, guinea pig inclusion conjuctivitis C. psittaci and feline conjuctivitis C. psittaci and may be correlated to a lack of antibody response to the chlamydial major outer membrane protein (MOMP) in these same strain/host combinations. This study also provides the first documented case of natural C. pneumoniae infection causing a severe and extended respiratory episode in a captive koala population. This captive episode is in contrast to most free-range observations in which koala C. pneumoniae is rarely documented as causing respiratory, ocular or urogenital tract disease.     

Modelling Chlamydia–koala interactions: coexistence, population dynamics and conservation implications

1. Considerable research has been conducted on koala Phascolarctos cinereus population dynamics and the epidemiology of Chlamydia psittaci infection in koalas, but the impact of Chlamydia on koala populations has been difficult to assess.
2. I developed a model of koala and Chlamydia population dynamics to examine interactions between Chlamydia transmission and pathogenicity, koala mating behaviour and demography, and koala population persistence.
3. Simulations based on sexual and parent–offspring parasite transmission demonstrate that stable Chlamydia–koala coexistence is possible in a small population for a broad range of demographic, behavioural, pathogenicity and transmission parameter estimations. Koala population persistence was most sensitive to reduced annual survivorship of adults (4–10-year-old males and 2–12-year-old females), highlighting the need for accurate field estimates of adult survivorship in order to assess Chlamydia 's impact on specific populations.
4. If koalas become less resistant to disease in fragmented, high-stress habitats (i.e. experience increased Chlamydia -induced mortality and sterility rates), Chlamydia is not predicted to cause koala extinctions under most conditions. Extinctions are only predicted if Chlamydia transmission rates also increase (e.g. due to new transmission pathways or increased mating frequency), or other non-disease factors change birth and mortality rates to reduce the koala population's intrinsic rate of increase below 0·1.
5. The most important predicted effect of habitat fragmentation and other forms of human disturbance on this unique host–parasite relationship is the extinction of Chlamydia in populations where koala resistance to disease decreases.     

衣原体质粒的研究进展

衣原体质粒是一个分子量约为7.5 kb,基因序列高度保守,非整合性的DNA分子,广泛存在于沙眼衣原体的各个血清型中,鼠衣原体和鹦鹉热衣原体也携带该质粒.近年来,人们发现衣原体质粒是一种毒力因子,可以导致小鼠输卵管积水.动物实验显示质粒缺失株可作为减毒活疫苗来预防衣原体感染所致的生殖道和眼睛的病变.不仅如此,衣原体质粒还是一种有效的基因操纵工具,可用于沙眼衣原体致病机制的研究.因此,开展对衣原体质粒的研究具有重要的意义.     

Abortion due to infection with Chlamydia psittaci in a sheep farmer's wife.

A farmer''s wife who had helped with lambing aborted spontaneously in March after a short febrile illness in the 28th week of her pregnancy. She developed disseminated intravascular coagulation post partum with acute renal failure and pulmonary oedema. Recovery was complete after two weeks of hospital care. A strain of Chlamydia psittaci, probably of ovine origin, was isolated from the placenta and fetus. The patient''s serum showed rising titres of antibody against chlamydia group antigen; the placental and fetal isolates; and a known ovine abortion, but not a known avian, strain of C psittaci. IgG against both ovine abortion and enteric strains of C psittaci was detected, but IgM against only an abortion strain was detected. Histological examination showed pronounced intervillus placentitis with chlamydial inclusions in the trophoblast but no evidence of fetal infection or amnionitis. Laboratory evidence of chlamydial infection was found in an aborting ewe on the farm in January and in remaining sheep and lambs in July. Doctors should recognise the possible risk to pregnant women in rural areas where chlamydial infections in farm animals are widespread.     

Chlamydiosis: seroepidemiologic survey in a red deer (Cervus elaphus) population in Italy

Chlamydiae are obligate, intracellular, gram-negative bacteria that are responsible for important diseases in humans, other mammals, and birds. Studies have shown that chlamydiae could be present in wild ruminants, but the serodiagnostic method most commonly used did not allow identification of chlamydial species. We determined the prevalence of antibodies to Chlamydia pecorum, Chlamydia suis, Chlamydia abortus, and Chlamydia psittaci in 271 red deer (Cervus elaphus) of a central Italian population, by using the microimmunofluorescence test that shows antibody response against genus-specific and species-specific antigens. No sera had detectable antibodies to C. pecorum and C. abortus. Antibodies were detected against C. psittaci (9.6%) and C. suis (3.3%). Antibody response could be related to contact of the red deer with birds and wild boars (Sus scrofa), respectively, and confirm an extended host range of individual Chlamydia species. In view of the potential zoonotic risk related to exposition of C. psittaci, our findings suggest surveillance of wild ruminants as potential reservoirs for chlamydiae.     

Detection of Chlamydia psittaci by Immunofluorescence

A direct fluorescent-antibody (FA) test was developed to detect Chlamydia psittaci in dural impressions from specimen-inoculated mice. Technical procedures for the test were compared. C. psittaci was found in mice after infection as early by the FA technique as it was by cytochemical staining methods usually used. The lymphogranuloma venereum organism was also stained by conjugated antibody to C. psittaci. A distinctive advantage of the described FA test is that organisms are identified immunologically as members of the genus Chlamydia simultaneously with their detection.     

In vitro expression of factor-mediated cytotoxic activity generated during the immune response to Chlamydia in the mouse

Supernatant fluid (SF) prepared by mitogen incubation of spleen cells from A/J mice previously immunized against lethal challenge by the 6BC strain of Chlamydia psittaci was cytotoxic for mouse fibroblasts (L cells) infected with 6BC, as detected by the [3H]thymidine release assay and the trypan blue exclusion test. In contrast, SF prepared from spleen cells taken from unimmunized animals (controls) was not cytotoxic when added to infected L cells. No cytotoxicity was observed when SF was added to uninfected L cells. Maximal levels of cytotoxicity were observed only from cells infected with 6BC for at least 26 hr and exposed to SF for greater than 20 hr. Furthermore, the degree of cytotoxicity was dependent on both the dose of Chlamydia administered and the concentration of SF in the medium. We conclude that the capacity to secrete a spleen cell cytotoxic factor is an aspect of the immune response against the obligate intracellular prokaryotic pathogen Chlamydia. Our results indicate that SF-mediated cytotoxicity is induced subsequent to immunization with Chlamydia, and is significantly more pronounced against infected as opposed to uninfected L cells.     

Newcastle disease virus and Chlamydia psittaci in free-living raptors from eastern Germany

Organ samples from free-living raptors from the federal states of Berlin and Brandenburg in eastern Germany were tested for Newcastle disease virus (NDV; n = 331) and Chlamydia psittaci (n = 39) by polymerase chain reaction (PCR). In 18 individuals NDV nucleic acids were detected. These samples originated from barn owls (Tyto alba; n = 15, 28%), tawny owl (Strix aluco; n = 1, 5%), common buzzard (Buteo buteo, n = 1, 1%), and European kestrel (Falco tinnunculus; n = 1, 4%). In 29 (74%) of 39 samples C. psittaci was detected. Chlamydia psittaci is common in free-living birds of prey in the investigated area.     

Plasmids of Chlamydia psittaci: Cloning and comparison of isolates by Southern hybridisation

Abstract A chlamydial plasmid, 6.2 kb in size, was isolated from an avian strain of Chlamydia psittaci and cloned into the Eco RI site of pUC13. A restriction enzyme cleavage map of the resultant clone, pAP¹p, was very similar to the published map of the plasmid cloned from the C. psittaci meningopneumonitis strain Cal-10. Southern hybridisation analyses using pAP¹p as a probe, revealed the presence of plasmids with homologous DNA sequences in avian psittacosis, avian ornithosis, ovine polyarthritis and sporadic bovine encephalomyelitis strains of C. psittaci , as well as the LGV strain of Chlamydia trachomatis . Plasmid was not detected in koala conjunctivitis, ovine abortion or feline conjunctivitis isolates. The plasmid-containing isolates could be grouped according to size (6.2 or 7.2–7.3 kb) and restriction endonuclease pattern. These three plasmid categories correlate with previously reported C. psittaci biotypes, immunotypes and serotypes. The absence of plasmid from three infectious, pathogenic strains of C. psittaci suggests that, in this species at least, plasmid-encoded genes are not essential for survival, infectivity or virulence of the parasite.     

Comparative PCR-based restriction fragment length polymorphism analysis of the plasmid gene orf3 of Chlamydia trachomatis and Chlamydia psittaci

The BfaI digestion of PCR-based restriction fragment length polymorphism analysis of the plasmid orf3 of Chlamydia trachomatis and Chlamydia psittaci provided evidence for two distinct restriction patterns, respectively. The nucleotide sequences of orf3 genes confirmed these differences. Serum antibodies against recombinant C. psittaci protein (pgp3) encoded by orf3 were detected both in pigeons with C. psittaci infection and in a human patient with psittacosis.     

Guineapig inclusion conjunctivitis (GPIC) in a commercial colony

Serological findings in a commercial colony of Hartley guineapigs revealed that about 70% had antibodies to Chlamydia psittaci as detected by the microimmunofluorescence method. Conjunctivitis was evident in 14% of 86 guineapigs examined. Chlamydial antigen was detected in conjunctival scrapings by a direct immunofluorescence test using Chlamydia-specific monoclonal antibody; however, C. psittaci was not demonstrated by other methods.     

Seroepizootiology of selected infectious disease agents in free-living birds of prey in Germany

Four hundred forty-eight blood plasma samples from free-living birds of prey from Berlin and the Brandenburg area in eastern Germany were tested for antibodies against Newcastle disease virus (NDV), falcon herpesvirus (FHV), owl herpesvirus (OHV), and Chlamydia psittaci. Antibodies to NDV were detected in 6 (2%) of 346 tested diurnal birds of prey, whereas none of the owls (n = 55) was positive. The positive samples originated from two common buzzards (Buteo buteo), three ospreys (Pandion haliactus) and one marsh harrier (Circus aeruginosus). Titers varied between 1:8 and 1:32. Of 253 birds of prey one osprey (<1%) tested positive for antibodies to FHV with low titer of 1:6. This is the first detection of antibodies against FHV in an osprey. Furthermore, antibodies against OHV could be found in one tawny owl (Strix aluco) and one common buzzard (2 of 253, 1%) with low titers of 1:6. Of 422 birds of prey 267 (63%) tested positive for antibodies to Chlamydia psittaci with titers varying between 1:5 and 1:256 which reflects the ubiquitous occurrence of Chlamydia psittaci in these birds of prey.     

Inbreeding and disease avoidance in a free‐ranging koala population

Habitat destruction and fragmentation are increasing globally, forcing surviving species into small, isolated populations. Isolated populations typically experience heightened inbreeding risk and associated inbreeding depression and population decline; although individuals in these populations may mitigate these risks through inbreeding avoidance strategies. For koalas, as dietary specialists already under threat in the northern parts of their range, increased habitat fragmentation and associated inbreeding costs are of great conservation concern. Koalas are known to display passive inbreeding avoidance through sex‐biased dispersal, although population isolation will reduce dispersal pathways. We tested whether free‐ranging koalas display active inbreeding avoidance behaviours. We used VHF tracking data, parentage reconstruction, and veterinary examination results to test whether free‐ranging female koalas avoid mating with (a) more closely related males; and (b) males infected with sexually transmitted Chlamydia pecorum. We found no evidence that female koalas avoid mating with relatively more related available mates. In fact, as the relatedness of potential mates increases, so did inbreeding events. We also found no evidence that female koalas can avoid mating with males infected with C. pecorum. The absence of active inbreeding avoidance mechanisms in koalas is concerning from a conservation perspective, as small, isolated populations may be at even higher risk of inbreeding depression than expected. At risk koala populations may require urgent conservation interventions to augment gene flow and reduce inbreeding risks. Similarly, if koalas are not avoiding mating with individuals with chlamydial disease, populations may be at higher risk from disease than anticipated, further impacting population viability.     

Interspecies structural diversity among chlamydial genes encoding histone H1.

Recently, a eukaryotic histone H1-like protein has been detected in Chlamydia trachomatis serovar L2 [Hackstadt et al., Proc. Natl. Acad. Sci. USA 88 (1991) 3937-3941; Tao et al., J. Bacteriol. 173 (1991) 2818-2822]. We have cloned the corresponding gene from C. trachomatis serovar J and the Chlamydia psittaci strain mn. Sequencing demonstrated absolute gene identity between the two C. trachomatis serovars L2 and J, but divergence in the C. psittaci strain mn. These differences resulted in altered aa residues (in particular no cysteines) and a smaller molecular mass for H1 from C. psittaci strain mn. The amino acid (aa) sequence comparisons with other histone proteins show best alignment to sea urchin H1, notably in the C terminus, for both C. trachomatis and C. psittaci histones. Chlamydial interspecies aa homology, however, is most conserved at the N terminus, suggestive of a bi-functional role for these unique histone proteins.     

Chlamydia psittaci ewe abortion agent: complete nucleotide sequence of the major outer membrane protein gene

The complete nucleotide sequence encoding the major outer membrane protein (MOMP) of Chlamydia psittaci strain A22/M, responsible for enzootic abortion of ewes (EAE), has been determined. An 800bp Eco RI/ Xba I fragment containing a portion of the MOMP coding sequence from C. trachomatis serovar L1 was used to probe a λL47.1 genomic library constructed from DNA obtained from C. psittaci EAE A22/M. The recombinant L47.1/EA1 was selected and contained the entire C. psittaci MOMP gene within a 7.5 kb Bam HI fragment. The DNA sequence revealed an open reading frame encoding 402 amino acids, including a 22 amino acid signal peptide, which exhibited 17/22 conservation with the signal peptide of C. trachomatis MOMP. The calculated molecular mass of the C. psittaci MOMP was 43 kDa. A comparison of the MOMP genes of C. psittaci and C. trachomatis revealed only 34% nucleotide sequence homology, but 65% amino acid homology.