A rapid monoclonal immunofluorescence assay for Chlamydia psittaci in fecal smears from psittacine birds

One hundred two fecal specimens from psittacine birds submitted to Veterinary Laboratory Services of the California Department of Food and Agriculture at Petaluma were screened for Chlamydia psittaci by a direct immunofluorescence assay using a fluorescein-labeled monoclonal antibody conjugate specific for Chlamydia sp. Results were compared with those obtained by isolation of chlamydia in cultures of McCoy mouse cells. The relative specificity of the direct fluorescent antibody test on fecal smears was 98.9% and the relative sensitivity was 62.5%. The results of this study suggested that the direct fluorescent antibody test was highly specific, and it proved to be a useful same-day antemortem diagnostic test for birds with symptomatic chlamydial infection. The use of centrifugation in the cell culture assay was found to significantly enhance the level of chlamydial infection in cell culture.     

Direct immunofluorescence tests with counterstains for detection of Chlamydia psittaci in infected avian tissues.

Different methods of preparation and serological evaluation of rabbit globulins for use in fluorescent antibody conjugate and different methods of counterstaining with fluorescent antibody tests were evaluated for detection of Chlamydia psittaci in infected turkey tissues. The agar gel precipitin reaction was that chosen for testing and selecting antiserums to be used for fluorescein isothiocyanate conjugation. The fluorescent antibody staining was most pronounced with conjugate made from globulins precipitated with ammonium sulfate. A direct fluorescent antibody method with Evans blue counterstain correctly identified "coded" specimens of C. psittaci-infected and noninfected turkey air sacs. However, naphthalene black was superior to Evans blue as a counterstain when infected pericardial sacs were tested.     

Isolation of Chlamydia psittaci from nasal and conjunctival exudate of a domestic cat

A feline strain of Chlamydia psittaci was isolated in tissue culture from nasal and conjunctival swabs from a free range domestic cat with bilateral conjunctivitis and rhinitis, living in the Liverpool area of the UK. The isolate was identified as C psittaci on the basis of its characteristic inclusion morphology in cell culture and by means of specific indirect immunofluorescence with known C psittaci specific antiserum. The isolate could be differentiated from other chlamydiae of non-feline origin by its amino acid nutritional requirements.     

Evaluation of a monoclonal antibody based ELISA for detection of feline Chlamydia psittaci

A commercially available ELISA designed for the detection of C trachomatis in human urogenital specimens was compared with cell culture for the detection of Chlamydia psittaci in cat conjunctival swabs and in twofold dilutions of a cell culture pool of a feline strain of C psittaci. Cell culture was more sensitive than the ELISA test for detection of C psittaci.     

Evaluation of an enzyme immunoassay for detection of Chlamydia psittaci in vaginal secretions, placentas, and fetal tissues from aborting ewes

A commercially available enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in human urogenital and conjunctival specimens was compared with isolation in cell culture for the detection of Chlamydia psittaci in vaginal and placental swabs from aborting ewes and swabs of aborted fetal tissues. The EIA on vaginal swabs collected from 10 ewes experimentally infected with C. psittaci had a sensitivity of 85.7% and a specificity of 85.7%. Vaginal swabs collected at the time of abortion or within 3 days were the best samples for detection of chlamydial infection. The 29 vaginal swabs collected during this period from experimentally infected ewes were all strongly EIA-positive, and chlamydia were isolated from 28. The EIA on vaginal swabs from 78 field cases of abortion had a sensitivity of 78.0% and a specificity of 76.8%. The EIA on swabs of cotyledons from 65 placentas had a sensitivity of 100% and a specificity of 75.0% compared with isolation in cell culture. The EIA on 57 swabs of fetal tissues or body fluids from 10 aborted fetuses or weak lambs from experimentally infected ewes had a sensitivity of 26.6% and a specificity of 88.1% compared with isolation in cell culture. Limitations of the EIA are discussed.     

Comparison of the omp I gene of Chlamydia psittaci between isolates in Victorian koalas and other animal species

Objective The objective of this study is to compare the strain of chlamydia causing genital infection in koalas from Victoria with isolates from other animal species.
Design Polymerase chain reaction and restriction enzyme analysis has been used to compare various Chlamydia psittaci isolates from a range of animals and disease syndromes. The isolates used in this study include isolates from three birds, three from aborted sheep, one from polyarthritis, one from bovine abortion, one from feline pneumonitis, three porcine isolates from faeces, polyarthritis and abortion, and three urogenital isolates from Victorian koalas.
Procedure Two polymerase chain reactions were performed, each amplifying a different region of the omp I gene. The first polymerase chain reaction amplified a 144 bp segment of the gene which was then digested with the restriction enzyme Eco R I. The second polymerase chain reaction amplified a larger 1070 bp region of the omp I gene which was digested with two restriction enzymes Alu I and Nde II.
Results and conclusions The results obtained have confirmed that variation in DNA sequence of various animal chlamydia isolates does occur. They have also shown that it is possible to classify isolates, based on their restriction enzyme profiles, into distinct groups.     

Polymerase chain reaction for detection of Ureaplasma diversum from urogenital swabs in cattle in Australia

Background Ureaplasma diversum has been associated with various reproductive problems in cattle, including granular vulvovaginitis, endometritis, salpingitis, early embryonic death, weak calves, decreased conception rates, balanoprosthitis, impaired spermatozoids and seminal vesiculitis in bulls. Methods This study briefly outlines the use of polymerase chain reaction (PCR) for the rapid detection of U. diversum directly from urogenital swabs collected from Australian beef cattle. Results The 16S ribosomal RNA gene sequences obtained from the PCR products of the clinical samples were closely related to U. diversum strain A417. Conclusion The present test enabled detection of the organism directly from clinical swabs collected from animals with or without lesions.     

Rapid detection of Chlamydia psittaci in vaginal swabs of aborted ewes and goats by enzyme linked immunosorbent assay (ELISA)

An enzyme linked immunosorbent assay (ELISA) for the detection of Chlamydia psittaci in vaginal swabs of aborted ewes and goats has been developed using microtiter plates coated with sheep anti-Chlamydia immunoglobulin G. This technique was compared to the direct isolation of the agent by plaque assay on McCoy cells. Among 89 specimens from animals in infected flocks, 58 were positive by both methods, seven were only positive by ELISA, and nine others were only positive by direct isolation (plaque assay). None of the 75 specimens from animals in healthy flocks gave a positive response in ELISA or the plaque assay. Unlike direct isolation in cell culture, the ELISA technique permitted the detection of Chlamydia even in the absence of special care in sampling and conservation of specimens.     

Evaluation of the polymerase chain reaction in comparison with other diagnostic methods for the detection of Chlamydia psittaci.

Various diagnostic methods exist for the detection of Chlamydia psittaci. In the current study, the test performance of polymerase chain reaction (PCR) was compared with other testing methods used in the diagnosis of C. psittaci. Tissue and fecal specimens (n = 119) of avian and mammalian origin were tested by PCR and one or more of the following methods: cell culture, enzyme-linked immunosorbent assay, and direct fluorescein-conjugated monoclonal antibody staining. Several gold standards, based on results of testing methods other than PCR, were used to calculate the following test performance characteristics of PCR: sensitivity and specificity, with their 95% confidence intervals; kappa statistics, a measure of intertest agreement; and lambda statistics, a chance-corrected estimate of the sensitivity and specificity. Overall, the test performance characteristics of PCR were low compared with the other testing methods. Possible reasons for the poor test performance of PCR in the current study include destruction of the organisms during storage, interference with the PCR by other reagents, or technical errors.     

Immunocytologic detection of Chlamydia psittaci from cervical and vaginal samples of chronically infected ewes.

An immunocytologic method was developed for the detection of chronic Chlamydia psittaci infection from the reproductive tract of ewes. Vaginal and cervical samples from 8 infected and 2 non-infected ewes were stained with a C. psittaci-specific monoclonal antibody. Cells containing C. psittaci were only detected from the 8 infected ewes and the level of detection varied with respect to the estrus cycle. An increased number of infected cells were observed during the periovulation period, thus indicating an optimal window for detection.     

Simple transport medium for the isolation of Chlamydia psittaci from clinical material

Infectious elementary bodies of Chlamydia psittaci in tissue samples from field cases of enzootic abortion were placed in five different transport media (A to E). In one medium, in the absence of refrigerative storage, the organism remained viable for 30 days and at 4 degrees C for 34 days. This was medium D; it consisted of sucrose (74.6 g/litre), K2HPO4 (1.237 g/litre), L-glutamic acid (0.721 g/litre), fetal calf serum (10 per cent v/v), vancomycin and streptomycin (100 micrograms/ml) and nystatin and gentamicin (50 micrograms/ml). Samples of this transport medium were supplied to veterinary investigation centres throughout the UK. Of 1862 samples submitted for diagnosis of enzootic abortion only 1.55 per cent were so contaminated that chlamydiae could not be detected. This transport medium permits the isolation of C psittaci from clinical material for up to about one month, even in the absence of conventional storage facilities.     

Experimental conjunctival infection of lambs with a strain of Chlamydia psittaci isolated from the eyes of a sheep naturally affected with keratoconjunctivitis

Five ram-lambs were inoculated into the left conjunctival sac with the 15R isolate of Chlamydia psittaci, recovered from a sheep with keratoconjunctivitis. A sixth ram-lamb was kept in contact with them. The five lambs developed varying degrees of acute conjunctivitis and 14 days later C psittaci could be recovered from the inoculated eyes, from which Branhamella ovis was also isolated. The eyes were examined regularly for four months; C psittaci could not be re-isolated but the eyes developed varying degrees of follicular conjunctivitis. After four months the sheep were treated with corticosteroids in an attempt to reactivate a latent chlamydial infection but no chlamydiae could be isolated. Five months after the start of the experiment the six lambs were inoculated with 15R into the left conjunctival sacs. Acute conjunctivitis developed which was not as severe as after the first inoculation, but C psittaci could only be recovered from the left eyes of three sheep three days after inoculation. The eyes remained chronically affected by follicular conjunctivitis. Six months after the start of the experiment the left eyes were again inoculated with 15R; on this occasion acute conjunctivitis did not develop and chlamydiae could not be isolated. Chronic follicular conjunctivitis persisted until the experiment was terminated three months later.     

Chlamydia psittaci latex agglutination antigen for rapid detection of antibody activity in avian sera: comparison with direct complement fixation and isolation results

Cell-culture-grown Chlamydia psittaci of turkey origin was treated with phenol and partially purified by differential centrifugation. The most stable antigen/latex mixture occurred with crude antigen precipitated by dimethyl sulfoxide and digested with trypsin. Agglutination reactions occurred within 2 minutes when antigen/latex and antibody-positive serum mixtures were rotated to facilitate contact of the reagents. Nonspecific agglutination of control latex occurred with 1.2% of clinical sera. When C. psittaci was isolated from feces from individual birds, latex agglutination (LA) and direct complement fixation (DCF) together detected antibody activity (titers greater than or equal to 8) in significantly more cases than did DCF alone. It follows, then, that an LA titer alone is highly indicative of a currently active infection or one that has occurred only recently. The isolation rate from birds that had no antibody activity detectable by LA or DCF was 3.8%. In tests on paired clinical sera, LA and DCF agreed in 72.5% of the cases regarding increased, decreased, or stable titers. For detection of antibody activity in single sera, LA had a sensitivity of 39.1% and a specificity of 98.8% relative to DCF detection of antibody activity. The high specificity corroborates the usefulness of LA as an indicator of current or very recent infection.     

Evaluation of bioaerosol sampling techniques for the detection of Chlamydophila psittaci in contaminated air

Chlamydophila (C.) psittaci, a category B bioterrorism agent, causes respiratory disease in birds and psittacosis or parrot fever in man. The disease spreads aerogenically and no vaccines are available for either birds or man. Highly sensitive C. psittaci bioaerosol monitoring methods are unavailable. We evaluated: (1) dry filtration for collecting C. psittaci from contaminated air using different samplers and membrane filters, (2) impingement into different liquid collection media by use of the AGI-30 impinger and the BioSampler and (3) impaction into newly designed C. psittaci media utilizing the MAS-100 aerosol impactor. For personal bioaerosol sampling, we recommend the use of a gelatin filter in combination with the IOM inhalable dust sampler at an airflow rate of 2L/min. This allowed the detection of 10 organisms of C. psittaci by both PCR and culture. For stationary bioaerosol monitoring, sampling 1000L of air in 10min with the MAS-100 impactor and ChlamyTrap 1 impaction medium was most efficient and made it possible to detect 1 and 10 C. psittaci organisms by PCR and culture, respectively. ChlamyTrap 1 in combination with the MAS-100 impactor might also be applicable for bioaerosol monitoring of viruses.     

高致病性猪蓝耳病直接免疫荧光诊断方法的建立及其应用

以临床"高热综合征"病例中分离的高致病性猪繁殖与呼吸综合征病毒(PRRSV)HBR变异株接种试验猪制备抗血清,采用硫酸铵盐析法提取血清中免疫球蛋白(IgG),并用异硫氰酸荧光素(FITC)对IgG进行荧光标记,建立了一种直接免疫荧光诊断方法(FA),用于检测病毒感染单层细胞及动物脏器组织切片中PRRSV抗原.本试验对PRRSV感染MARC-145细胞增殖动力学检测结果表明,接种后16 h可检测到抗原阳性细胞,接种后60 h～72 h达到病毒增殖高峰;对病毒人工感染猪脏器组织抗原分布检测结果显示,腹股沟淋巴结、空肠、睾丸、脾脏、下颌淋巴结、肠系膜淋巴结和扁桃体抗原阳性检测率较高;对临床10个猪场送检的38份病料平均阳性检出率为63.2%(24/38).该方法具有特异性强、敏感性和重复性好等特点,与RT-PCR检测结果符合率为96.3%,对几种已知猪病毒抗原无交叉反应,标记的荧光抗体于4℃保存6个月稳定.FA为PRRS的临床诊断提供了一种快速、便捷、敏感、特异的检测方法.     

A modification of the indirect immunofluorescence test for detection of Ehrlichia phagocytophila antibodies.

A modified technique for production of antigen and performance of the test is described. A suspension of infected neutrophils was directly applied to multiwell slides. Multichannel pipettes may be used for dilution and application of sera. The modification increases the capacity both by production of the antigen and by performance of the test. This paper also gives a quantitative determination of the antibodies.     

Indirect immunofluorescence test using polyclonal antibodies for the detection of Taylorella equigenitalis

Contagious equine metritis is a horse disease that causes endometrial inflammation due to Taylorella equigenitalis. Since Taylorella asinigenitalis was characterized, genital swab culture has proved to be an insufficient method for distinguishing between the two Taylorella species. Here, we developed an indirect immunofluorescence (IIF) test using polyclonal antibodies. Specificity, sensitivity, and detection limit were assessed using isolated bacteria (55 T. equigenitalis strains, 46 T. asinigenitalis strains and 18 other bacterial species), experimental and genital swabs in comparison to bacterial culture and polymerase chain reaction (PCR) testing. Our results indicated that IIF using polyclonal antibodies allows T. equigenitalis to be discriminated from T. asinigenitalis. This test constitutes a rapid, sensitive and specific tool for confirming presumptive colonies of T. equigenitalis.