Using quantitative polymerase chain reaction to correlate Chlamydia pecorum infectious load with ocular, urinary and reproductive tract disease in the koala (Phascolarctos cinereus)

Complex interactions between Chlamydia pecorum infection, the immune response and disease exist in the koala. We used quantitative polymerase chain reaction to investigate the relationship between C. pecorum infectious load and ocular and urogenital tract disease. Chlamydia pecorum shedding was generally higher in animals with chronic, active disease than in animals with inactive disease. The absence of ocular disease was generally associated with low levels of shedding, but relatively high levels of shedding in the urogenital tract were detected in some koalas without clinical disease signs. These results suggest a complex disease pathogenesis and clinical course in C. pecorum-infected koalas.     

Proctitis associated with chlamydial infection in a koala

Objective To describe proctitis associated with chlamydial infection in a koala.
Design A pathological study  

Animal

A free living, male koala aged 17 years.
Procedure Rectum was examined histologically and chlamydial organisms visualised using Giminez stain and an immunoperoxidase staining method using an anti- Chlamydia lipopolysaccharide (genus specific) antibody.
Results An aged koala presented for euthanasia was found to have asymptomatic chronic proctitis, cystitis, prostatitis, urethritis and conjunctivitis associated with chlamydial infection. Inflammation was severe in the terminal rectum and extended into the proximal common vestibule. Chlamydial organisms were visualised in the rectal surface epithelium using Giminez stain and an immunoperoxidase staining method. Organisms were also detected in the epithelium of the bladder, prostate and urethra.  

Clinical Implications

Possible modes of transmission for the rectal infection are direct sexual transmission or ascending infection by organisms shed from the urogenital tract into the common vestibule. Previously unreported chlamydial proctitis in the koala may represent a potential reservoir of infection for other koalas.     

Chlamydial infection in a colony of captive koalas

Forty three koalas in a captive colony were investigated for the presence of Chlamydia psittaci infection and associated disease. Swabs were taken from conjunctivae and urogenital sites for cell culture isolation of C psittaci and for cytological examination (direct smears) for chlamydial inclusions and evidence of inflammation. On the basis of cell culture isolation, 28 samples from 25 koalas were positive for C psittaci (that is, infected). Three koalas were positive from both sites, 5 from conjunctivae alone and 17 from urogenital sites alone. Seven of the 8 koalas with positive conjunctival swabs had overt signs of conjunctivitis, but only 3 of the 20 koalas with positive urogenital swabs had overt signs of 'wet bottom' (continual urine soiling due to cystitis) or purulent discharge. However, 5 of the 20 with positive urogenital swabs had past episodes of 'wet bottom'. Moreover, examination of direct cytological smears showed evidence of inflammation (neutrophils) in 7 of 8 koalas with positive conjunctival swabs and 17 of 20 with positive urogenital swabs. Chlamydial inclusions were rarely identified with surety on direct cytological smears. In the 18 koalas without chlamydia, one had overt conjunctivitis while 2 had past episodes of conjunctivitis. The koala with conjunctivitis at the time of sampling had a prior history of 'wet bottom'. Examination of direct cytological smears revealed 2 of the chlamydial negative koalas had high numbers of neutrophils in urogenital smears. It was concluded that C psittaci infection may cause overt or sub-clinical disease, with the former developing when the koalas were stressed through management procedures or concomitant disease.     

Epizootiology of Chlamydia infections in two free-range koala populations

The prevalence of Chlamydia pecorum and Chlamydia pneumoniae infections in two free-range koala populations was assessed using genus-specific PCR combined with species-specific DNA probe hybridisation. Population A had a very high overall level of chlamydial infection (85%) with significantly more of these infections being due to C. pecorum (73%) compared to C. pneumoniae (24%). The second population had a much lower prevalence of infection (10%) with equal levels of both species. An important finding of this study was that. while five of 24 C. pecorum-infected koalas had clinical signs of the disease (both ocular and urogenital sites), none out of seven C. pneumoniae-infected koalas had signs of clinical disease. This suggests that C. pecorum may be the more pathogenic of the two chlamydial species infecting this host. The level of infection (assessed by intensity of the specific hybridisation signal) also differed between chlamydial species, with C. pecorum infections ranging from low to high grade whereas C. pneumoniae infections were always low grade. When the age of infected koalas was examined, 58% of young, sexually immature koalas were found to have C. pecorum infections, increasing to 100% of koalas in the older age groups. This suggests that, in this population at least, young koalas are readily infected with C. pecorum from their mothers. While the infection levels with C. pneumoniae were too low to be statistically significant, again, sexually immature koalas were found to be infected. The recent separation of chlamydial infections in koalas into two species is beginning to indicate different epizootiologies for koala C. pecorum compared to koala C. pneumoniae.     

TRANSCUTANEOUS ULTRASONOGRAPHY OF THE AVIAN UROGENITAL TRACT

The purpose of this article is to provide a survey of the diagnostic use of transcutaneous sonographic examination of the urogenital tract of pet birds. Possibilities and limitations in imaging normal and altered components of this organ system are discussed. Ultrasonographic examination of the avian urogenital tract was evaluated in 386 pet birds. Abnormal clinical signs were present in 289 birds; 97 birds were normal. Transcutaneous ultrasonography proved to be a useful, noninvasive, and fast diagnostic tool, especially in imaging laminated eggs and changes of the oviduct. Until now, these pathologic changes were not diagnosable in birds with other common noninvasive methods (e.g. radiography). The imaging of low-grade changes of the oviduct, disorders of the kidney without enlargement of the organ, as well as roughening of the egg shell proved to be difficult. Sonographic imaging of neither the normal kidney nor the inactive gonads was possible.     

Evaluation of the Rabbit Urinary Tract

Urinary tract disease is not an uncommon presentation for pet rabbits. It is beneficial for the veterinarian treating these patients to have a thorough understanding of a rabbit's urogenital anatomy, renal physiology, and diseases that affect the kidney and urinary tract. The evaluation of the rabbit urinary tract requires a multidisciplinary approach including the procurement of a thorough history, signalment, assessment of clinical signs, physical examination, and diagnostic test results. To obtain a definitive disease diagnosis, more intensive diagnostic testing may be required, including contrast radiography, ultrasound evaluation, and histopathological assessment of biopsy samples. This article will provide information to aid in the diagnosis and treatment of rabbit urinary tract diseases.     

A mortality survey of free range koalas from the north coast of New South Wales

In the period 1980 to 1986, 127 free range koalas from the north coast of New South Wales were presented for necropsy. Thirty-three koalas had urogenital disease alone, 8 had respiratory disease alone, 2 had digestive tract disease alone, 12 had multiorgan disease, 48 had traumatic injuries, 6 had neoplasia, 3 had miscellaneous conditions, while 15 had no significant lesions. Common naturally occurring disease entities included cystitis, conjunctivitis, paraovarian cysts, metritis and pneumonia. Lymphosarcoma was the common neoplasm.     

Antibiotics for the preservation of koala (Phascolarctos cinereus) semen

Aim To determine the normal microbial flora of the koala ejaculate and prepuce in order to select appropriate antibiotics for addition into diluents designed for the preservation of semen.
Procedure Bacteriological samples of the koala prepuce (n = 12) and ejaculate (n = 20) were submitted for microbial culture and sensitivity testing. Microbial flora of ejaculates collected by electroejaculation and artificial vagina were compared. The effects of varying concentrations of penicillin G and gentamicin on sperm motility and on the growth of bacteria in diluted semen stored at room temperature and 16°C over a 24 h period were investigated.
Results A range of bacteria was isolated from the koala prepuce and ejaculate. The predominant organisms in semen collected by electroejaculation and artificial vagina were Corynebacterium spp, none of which could be assigned to any recognised species. The addition of penicillin G and gentamicin to a PBS-based diluent at dose rates of 1000 to 2000 IU/mL and 100 to 200 m g/mL respectively, resulted in no adverse effect on sperm motility over a 24 h incubation period. Penicillin G (1000 IU/mL) and gentamicin (100 m g/mL) prevented growth of bacterial contaminants in diluted koala semen.
Conclusion By controlling the growth of bacteria in extended koala semen, penicillin G and gentamicin are likely to lengthen the period by which spermatozoa can be stored at 16°C and reduce the possibility of disease transmission during artificial insemination procedures.     

Use of gray-scale ultrasonography in the diagnosis of reproductive disease in the bitch: 18 cases (1981-1984)

Gray-scale ultrasonography was utilized in addition to radiography in the diagnosis of reproductive disease in 18 bitches. In 72% of the cases, ultrasonography was considered diagnostic because it revealed information on organ architecture, relationships of radiographically silhouetting soft tissue structures, and fetal viability that was unobtainable by radiography alone. In the remainder of the cases, ultrasonography was contributory to the diagnostic process by supporting the clinical and radiographic diagnoses. The benefits of ultrasonography are discussed, as is the ultrasonographic appearance of a variety of reproductive tract diseases.     

The koala (Phascolarctos cinereus)! past, present and future.

The koala (Phascolarctos cinereus) (from the Greek phascolarctos meaning leather pouch and bear, cinereus ashen grey in colour) is Australia's most admired native animal but very little scientific reference material is available on this unique animal. The demands of the fur trade during the latter half of the last century and up to the 1920's almost exterminated the koala and this tragic episode from the past is briefly recounted. Koalas are nocturnal and arboreal. Details of appearance, distribution, breeding, diet, water intake, volatile eucalypt oil detoxication and handling are briefly described, while information concerning intravenous injections, anaesthetic agents antibiotic therapy and fluid therapy are given. As an aid to veterinarians presented with koalas to examine, tables are presented for physiological, haematological and biochemical values. Pathological conditions found on post mortem examination are summarised, while disease conditions such as anaemia, keratoconjuctivitis, toxoplasmosis, cryptococcocis, reproductive disorders, miscellaneous conditions and internal and external parasites are mentioned. Finally, the future of the koala is discussed. Diminishing habitat, depletion of food supplies, bushfires, the threat of disease, coupled with a paucity of relevant scientific information, create an uncertain future for the koala. A plea is made to totally protect and expand the very few remaining known koala breeding areas especially in Victoria, and for veterinarians to become involved in conservation programs and wildlife research in order that the future of the koala in common with all other native species may be assured.     

1例犬传染性性病肿瘤的诊治体会

犬感染传染性性病肿瘤(CTVT)可导致犬的泌尿生殖道的炎症、长期失血,严重影响犬只健康。介绍了1例犬传染性性病肿瘤的临床检查、诊断和治疗,以期为该病的防控提供参考。     

Ventral Approach to the Pelvic Canal in the Female Dog

A ventral approach to the pelvic canal by bilateral pubic and ischial osteotomy was performed in five female dogs. The entire urogenital tract could be exposed and easily manipulated through this approach. All animals were ambulatory within 24 hours. Clinical union had formed in three of the four osteotomies by the time of euthanasia (months 2-4) in all animals. Incomplete union in some osteotomies indicated that exercise should be restricted for at least 4 months after the procedure. The procedure was determined to be clinically useful when wide exposure of the entire female urogenital tract is desired.     

Assessment of the urinary system.

History and physical examination alone will most often provide conclusive evidence of urinary tract disorders in ruminants. Detailed examination of the urinary system is described, and examples of disease are presented. Ancillary testing may be indicated to confirm the diagnosis, direct treatment, or clarify the prognosis. Serum chemistry interpretation, urinalysis, urinary tract endoscopy and radiography, ultrasonography, abdominocentesis, and renal biopsy are discussed.     

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.     

Further characterisation of the immune response of the koala

Sensitive enzyme immunoassays (EIA) were developed to monitor antibody (Ab) production in the koala, in response to both soluble and particulate antigens (Ag). When compared with a eutherian mammal, the rabbit, both the dynamics and kinetics of Ab production in the koala were found to be severely retarded. In vitro, Ag specific lymphocyte proliferative responses were demonstrated for the first time in this animal by sensitising koalas in vivo with Bacillus Calmet-Guerin (BCG), with the level and timing of this cell mediated immune (CMI) response comparable with those seen in non-metatherian mammals. Levels of circulating B lymphocytes were examined in an attempt to clarify the retarded humoral responses to foreign Ags. In addition, peripheral mononuclear cells (PMC) from koalas, were examined for their reactivity to a range of monoclonal Abs and lectins in an attempt to characterise these cells further. The lectins examined, demonstrated an all or none reactivity with koala lymphocytes and were therefore considered unsuitable as markers for identifying lymphoid subsets in this animal. A monoclonal Ab directed at class II MHC Ags in the mouse, demonstrated cross reactivity with a high percentage of all koala monocytes tested. Using this Ab to probe CMI responses in vitro, it is concluded that immune interactions required for such responses in the koala parallel those seen in other mammals.     

Purification and initial characterisation of koala immunoglobulins

The production of koala immunoglobulins (Ig) was elicited by the immunisation of a koala (Phascolarctos cinereus) with bovine serum albumin. This Ig was then purified using the highly specific techniques of affinity chromatography. The purified protein was compared with a "potential" koala Ig protein which was subsequently purified by Protein G chromatography and both proteins were further characterised by agarose/SDS-PAGE and immunoelectrophoresis. Results indicate that koalas do produce Ig in response to antigen challenge, koala Ig has a higher net negative charge than that seen in most other mammals and possibly two subclasses of IgG and IgM are present in normal koala serum.     

Urogenital problems in the neonatal foal

The common conditions affecting the urogenital tract of the neonatal foal and their medical and surgical management are described. Early recognition and treatment of serious abnormalities are necessary for a successful outcome in the equine neonate.     

Assessment of anti-bovine IL4 and IFN gamma antibodies to label IL4 and IFN gamma in lymphocytes of the koala and brushtail possum

We assess anti-bovine IL4 and IFN gamma (IFNg) antibodies for their ability to label IL4 and IFNg in koala (Phascolarctos cinereus), common brushtail possum (Trichosurus vulpecula) and mountain brushtail possum (Trichosurus caninus) lymphocytes using flow cytometry and immunohistochemistry to determine their applicability to studies of host response to intracellular pathogens. Anti-IFNg labelled a product of PMA-ionomycin stimulated sheep, koala and possum lymphocytes. High intensity labelling was not reduced by blocking non-specific binding with 10% FCS; and non-permeabilised koala lymphocytes labelled less, demonstrating that the labelled product was intracellular. The anti-IL4 antibody labelled variably more cells than the irrelevant antibody in some stimulated and non-stimulated preparations in all species but intensity of this labelling was similar to that of cells labelled with the irrelevant antibody. In this study, the antibodies did not label frozen or formalin-fixed tissues in a range of immunohistochemical techniques. We expect the anti-IFNg antibody to be effective in evaluating Th1 responses of koalas and possums exposed to various host, pathogen and environmental factors and add to the limited tools available for investigating the pathogenesis of marsupial diseases, especially those caused by intracellular organisms, such as tuberculosis of brushtail possums and chlamydial disease of koalas.     

Serological assessment of chlamydial infection in the koala by a slide EIA technique

A rapid and simplified slide enzyme immunosorbent assay (EIA) was developed for the diagnosis of chlamydial infection in the koala. HeLa 229 cells infected with koala strain Chlamydia psittaci were fixed on the surface of multiwell slides and used as the antigen. The assay consisted of first reacting koala antiserum with the fixed C psittaci antigen, followed by reaction with biotinylated rabbit anti-koala IgG, ABC reagent and substrate. The chlamydial EIA antibody titres obtained were compared with those of a complement fixation (CF) test using koala strain C psittaci as antigen. Of 35 koala sera tested, 16 CF positive sera (greater than or equal to 1:8) also had a positive titre (greater than or equal to 1:200) in the slide EIA test (sensitivity 93.8%, 15/16). Nineteen CF negative sera were also negative in the slide EIA (specificity 100%, 19/19). Sixty-eight samples of koala blood were collected by ear-prick using a sampling paper method and were assayed by both tests. Sensitivity of the slide EIA was 100% (15/15) and specificity of the test was 96.2% (51/53). To simplify the slide EIA for use as a practical screening test, a 3-point serum dilution series (1:100, 1:200, 1:400) was used. This 3-point slide EIA was compared with the CF test using sheep strain chlamydial antigen. Thirty-nine sera were assayed by both tests. The sensitivity of the 3-point method was 85.7% (6/7) and the specificity was 71.9% (23/32) as compared with the sheep antigen CF test.(ABSTRACT TRUNCATED AT 250 WORDS)