Single DNA sequence common to all chlamydial species employed for PCR detection of these organisms.

Chlamydial infection is responsible for a wide spectrum of diseases of the eye, genitourinary tract, and lung. This group of organisms is also implicated in the pathogenesis of coronary artery disease as well as arthritis. Since cross-species infection is widely reported (though probably underestimated), it is an advantage to have a rapid and reliable method to detect all forms of chlamydiae in patient samples. We have identified a 160/163-bp DNA fragment in Chlamydia which is highly conserved in all chlamydial species. A polymerase chain reaction method based on this sequence has been developed to detect, in clinical samples, chlamydiae which have been shown to be positive by fluorescent-staining immunoassay; this method can be utilized in combination with restriction endonuclease cleavage to identify individual chlamydial species. Thus we have developed a sensitive and rapid detection method and have used it on samples from patients with respiratory and genital infections.

Morphology of the thyroid in coastal and noncoastal populations of the koala (Phascolarctos cinereus) in Queensland.

The gross morphology, histology, and ultrastructure of the thyroid gland of the koala, Phascolarctos cinereus, is described. Generally, the glands were found to contain large-diameter follicles in association with an epithelium of low height. Morphometric analysis demonstrated a high relative thyroid weight (0.3 +/- 0.2 g/kg) for koalas compared with the 0.07-0.24 g/kg typical of eutherian mammals and 0.03-0.1 g/kg found in other marsupials. The relative thyroid weight of glands (0.33 +/- 0.21 g/kg) from the coastal population (less than 28 km from the coastline) was found to be significantly higher (ANOVA: P = 0.007, significant at the 1% level) than that for glands (0.21 +/- 0.11 g/kg) of noncoastal koalas (greater than 28 km from the coastline). Follicle size was positively correlated (at the 0.1% level) with relative thyroid weight in the overall koala sample. The presence of C cells, occurring singly in the epithelial layer, was demonstrated in electron micrographs. Structural features such as low epithelial height, large follicle length and width, and large intercellular spaces in association with low concentrations of free T4 (3.3 +/- 2.1 pM) and free T3 (1.4 +/- 0.9 pM) as reported previously (Lawson et al., 1996) are consistent with an unusually low level of glandular activity in the koala thyroid even though iodine concentrations in the thyroid gland [4.7 +/- 1.6 mg/g (dry weight)] as well as leaf [0.8 +/- 0.3 microg/g (dry weight)] and soil samples [3.8 microg/g (dry weight)] from the koalas' habitat appear unremarkable.

Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: cloning and characterization.

We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus). In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydia which are involved in the immunogenic response. Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C. psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain. The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an XbaI fragment, 2.4 kb in size (pKOC-5). DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C. pneumoniae, C. psittaci 6BC and C. trachomatis, and the kdtA gene of E. coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A. Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis. Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E. coli.

Concentrations of thyroid hormones and iodothyronine binding proteins in serum of the koala (Phascolarctos cinereus).

OBJECTIVE: To examine circulating total and free thyroid hormone (T3 and T4) concentrations, determine serum iodothyronine binding characteristics and estimate thyroid stimulating hormone (TSH) activity in sera of coastal and inland koalas. DESIGN: A prospective study. PROCEDURE: Koala serum T3 and T4 were measured by radioimmunoassay. T4 binding parameters were determined by radioligand binding and electrophoresis. Koala TSH values were determined by bioassay. RESULTS: Mean total T4 concentrations were 3.2 +/- 2.1 nM although values were significantly higher in inland-dwelling females in comparison to coastal-dwelling males. Free T4 was 3.3 +/- 2.1 pM. Total and free T3 were 0.4 +/- 0.2 nM and 1.4 +/- 0.9 pM respectively, although these values were at the lower end of the assay detection limit and should be viewed with reservation. Electrophoresis of [125I]-T4-labelled serum revealed only two proteins of electrophoretic mobility similar to human transthyretin (TTR) and albumin. Scatchard analysis of T4 binding to serum gave a curvilinear plot, which could be resolved into two binding sites with affinities identical to that of TTR and albumin but both of low concentration. The bioactivity of the TSH present in the sera was measured using a cell line (JP09) transfected with the human TSH receptor. The mean level of stimulation found in the sera corresponded to a bovine TSH activity of less than 10 mU/L. CONCLUSION: These results suggest that the serum concentrations of free and total thyroid hormones in koalas are low compared to other marsupials and very low compared to eutherian mammals. The mechanism of maintenance of euthyroidism in this species remains to be determined.

Seminal characteristics and spermatozoal morphology of captive Queensland koalas (Phascolarctos cinereus adustus).

Viable koala spermatozoa were successfully collected from 8 of 11 captive Queensland koalas on 52 of 76 attempts using electroejaculation under general anesthesia. Semen characteristics, including sperm concentration, percent progressive sperm motility and nuclear heterogeneity appeared to be similar to those of free-ranging Victoria koalas (P.c . victor). Two new head morphotypes (Type XI and teratoid) were identified, and the incidence of midpiece and principal piece spermatozoal abnormalities were recorded.

Remarkable sequence relatedness in the DNA encoding the major outer membrane protein of Chlamydia psittaci (koala type I) and Chlamydia pneumoniae.

DNA encoding the major outer membrane protein (MOMP) of the koala type-I strain of Chlamydia psittaci (pathogen responsible for blindness and infertility in koalas) was cloned and sequenced. Comparison with momp gene sequences from other chlamydial species revealed a remarkable degree of homology (> 97%) with that of the human pathogen, Chlamydia pneumoniae. In comparison, the sequence only shared 75% DNA sequence homology with other C. psittaci members and 69% homology with C. trachomatis. The open reading frame consisted of 1167 bp encoding a 389-amino acid (aa) pre-MOMP including a leader sequence of 23 aa, similar to the C. pneumoniae gene. These genes were closely related even within the four variable domains (86-100% homology). Specific antibodies were capable of distinguishing between koala type I and C. pneumoniae. This very high degree of relatedness between C. pneumoniae, a human pathogen, and an individual strain of C. psittaci in the momp gene raises further questions on the host specificity, classification and evolutionary relationships of the different chlamydial species.

Immuno-dot blot as a rapid diagnostic method for detection of chlamydial infection in koalas (Phasolarctos cinereus).

The sensitivity and specificity of an immunoscreening test for anti-chlamydial antibodies in koala (Phasolarctos cinereus) serum were determined after the adsorption of non-specific antibodies. The results of the test were compared with complement fixation tests, tissue culture, gene probe analysis and dot blot immunoscreening for host-borne chlamydial antigen. The immunoscreening test was the most sensitive test for the identification of chlamydial infection in koala serum samples, furthermore it was rapid, taking approximately 16 hours to complete, and inexpensive. However, for the assay of swab material from koalas, gene probe analysis remains the most sensitive method of detection of chlamydiae.

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

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.

Detection of Chlamydia psittaci in free-ranging koalas (Phascolarctos cinereus): DNA hybridization and immuno-slot blot analyses.

DNA-slot hybridization and immuno-slot blot analyses were compared for the detection of Chlamydia psittaci in crude swab material from free-ranging koalas. Immuno-slot blot analysis detected chlamydiae in 43 out of 68 koalas, with the sensitivity of the assay varying from 52 to 73% depending on the site of infection. Gene probe analysis was also used employing a genus-specific probe pCKO-10 isolated from a koala chlamydial gene library (ocular strain) and a plasmid probe pCKU cloned from a urogenital strain. The sensitivity of these two assays was comparable and they were considerably more efficient than the immuno-slot blot method for the detection of chlamydiae. Comparison of these data with a cell-culture method of detection, previously used with the same samples, demonstrated that gene probe analysis detected more positives than observed with cell culture. However, this appears to reflect more on the condition of the swab material rather than the sensitivity of the method.

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.

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.

Properties of equine luteinizing hormone alpha subunit alone and in combination with various beta subunits.

Previous studies have shown that equine luteinizing hormone (eLH) inhibits production of cyclic adenosine monophosphate (cAMP) induced by follicle-stimulating hormone (FSH) in preparations of seminiferous tubules from immature rats. It was also shown that the inhibitory effect was a function of the equine LH (eLH) alpha subunit. To explore this phenomenon further, the intrinsic FSH-like activities of eLH alpha alone and in combination with ovine (o) LH beta, ovine FSH beta, and equine FSH beta were evaluated in several assay systems. In a radioreceptor assay employing 125I-o-FSH and testis membranes from day-old calves, eLH was twice as active as oFSH, eLH alpha was 6% as active as oFSH, and other subunits showed a lack of activity (less than 1.5%). Whereas oLH was only 0.1% as active as oFSH, the hybrid eLH alpha-oLH beta was 3.0% as active. The binding activity of eLH alpha-FSH beta hybrids tended to be higher than the oFSH alpha-FSH beta hybrids. In the cAMP production assay, eLH alpha-FSH beta hybrids exhibited dampened dose-response curves when compared to the oFSH alpha-FSH beta hybrids. In a plasminogen activator assay (PAA) employing granulosa cells from intact 21-24-day-old female rats primed with diethylstilbestrol, eLH had activity comparable to that of oFSH, while eLH alpha was inactive. When eLH alpha was recombined with oFSH beta, eFSH beta, or oLH beta, the PAA stimulatory activity was not altered compared to that of the hybrids oLH alpha-oFSH beta, oFSH alpha-eFSH beta, and the recombinant oLH alpha-oLH beta, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The neurohypophysial hormones of the egg-laying mammals: identification of arginine vasopressin in the platypus (Ornithorhynchus anatinus).

Two neurohypophysial peptides have been purified from acetone desiccated posterior pituitary glands of the platypus (Ornithorhynchus anatinus) by molecular sieving and high-pressure liquid chromatography. A single pressor peptide, having an amino acid composition and a chromatographic retention time identical to those of arginine vasopressin, has been identified. A single oxytocic peptide has been isolated that ressembles oxytocin by its chromatographic retention time, but lack of material has prevented to obtain a correct amino acid composition. The pressor peptide is roughly four times more abundant than the oxytocic peptide. Neurohypophysial hormones of platypus seem similar to those of echidna, the other living prototherian, and to those of most placental mammals.

Aspects of the structure and development of monotreme spermatozoa and their relevance to the evolution of mammalian sperm morphology.

The elongated spermatid nuclei of monotremes exhibit a circumferentially arranged spiral pattern of chromatin condensation, and ultimately form helical filiform sperm heads up to 50 microns long and either circular or slightly oval in transverse section. The acrosome is formed by the collapse of the proacrosomal vacuole onto the rostral surface of the elongating nucleus. However, genesis of acrosomal material occurs in the absence of a prominent proacrosomal granule. The flagellum becomes inserted into the distal extremity of the nucleus, the most proximal mitochondria of the midpiece directly abutting the nuclear membrane, so that a prominent neck region is absent. The axoneme is simple and, in the midpiece, small dense peripheral fibres are closely applied to the outer surface of each of the nine microtubule doublets. The cortical fibrous sheath of the principal piece is an anastomosing spiral that lacks lateral columnar elements. The spermatozoal cytoplasmic droplet undergoes migration and is lost during epididymal passage. Monotreme spermatozoa exhibit a montage of features, some of these being also found in marsupials and some in sauropsidan vertebrates, as well as a number of their own distinctive features. It is concluded that monotreme spermatozoa also have a close affinity with the unspecialised spermatozoa of some eutherian mammals.