Preparation and use of a monoclonal antibody to detect Chlamydia psittaci antigen in paraffin-embedded tissue sections

A murine monoclonal antibody prepared against an ovine abortion isolate of Chlamydia psittaci (A22/Teramo) revealed specific binding to a 57 kDa chlamydial antigen in immunoblotting studies. The monoclonal antibody was able to detect intracytoplasmic chlamydial inclusions and scattered elementary bodies in infected McCoy cell culture, and on formalin-fixed paraffin-embedded tissue sections both from experimentally infected mice and from fetal membranes of cases of ovine enzootic abortion.     

High prevalence of chlamydial (Chlamydophila psittaci) infection in fetal membranes of aborted equine fetuses

Seventy-seven cases of equine abortion from 49 Hungarian farms that occurred between 1998 and 2000 were investigated for the presence of chlamydiae by immunohistochemistry, PCR and/or MZN staining. Evidence of the presence of these bacteria was obtained in 64 cases (83.1%) from 41 (83.7%) different farms. Partial ompA gene sequencing of PCR products revealed that the agent was Chlamydophila psittaci. Based on the findings of microbial diagnosis, pathology and case history, chlamydial infection was considered to be the most likely cause of abortion in at least 11 (14.3%) cases. In the remaining 53 Chlamydophila-positive cases, either other bacterial and viral agents (n = 22 or 28.6%) as well as non-infectious factors (n = 14 or 18.2%) were identified as more probable primary causes of disease, or the role of chlamydiae remained unclear because lesions in fetuses and fetal membranes were absent (n = 17 or 22.1%). When chlamydial antigen was detected in aborted equine placental tissue using immunohistochemistry it was seen only in the chorionic epithelial cells, but not in other parts of the fetal membranes nor in any of the fetal tissues. In conclusion, chlamydial infection of the genital tract should be considered a possible factor in equine reproductive disorders.     

Efficacy against ovine enzootic abortion of an experimental vaccine containing purified elementary bodies of Chlamydia psittaci

A vaccine prepared from purified, inactivated elementary bodies of Chlamydia psittaci protected sheep against abortion after subcutaneous challenge with live chlamydiae. Immunoblot analysis of serum samples revealed a consistently dominant antibody response against the chlamydial major outer membrane protein in all vaccinated sheep. Reactions to other chlamydial antigens were also detected but were less pronounced or inconsistent. Serological responses detected by complement fixation were variable and did not correlate with immunity.     

Chlamydia abortion in sheep: possibilities for serological diagnosis using a competitive ELISA and insight into the epidemiologic situation in Switzerland]

466 sheep sera out of 19 flocks in Switzerland were examined by a competitive enzyme linked immunosorbent assay (cELISA) for antibodies against Chlamydia psittaci "serotype 1" ("ovine enzootic abortion"). Since numerous positive reactors were found in flocks without abortion history, 30 fecal samples out of two of these flocks were examined by PCR for evidence of chlamydial DNA. One of these samples turned out to contain DNA of Chlamydia psittaci "serotype 1". These results suggest, that in Switzerland "serotype 1" of Chlamydia psittaci is widespread not only as cause of chlamydial abortion but also as latent intestinal infection in sheep. The resulting difficulties for serological diagnosis of chlamydial abortion and possible solutions based on the cELISA are discussed. The complement fixation test (CFT), still considered as standard method for serological examination for Chlamydiae, has additionally been applied.     

Chlamydia psittaci infection and associated infertility in sheep.

Nineteen ewes were injected subcutaneously with the agent of enzootic ovine abortion, Chlamydia psittaci serovar 1, at 50 days gestation. Placental and fetal tissues were examined at 15 days postinfection and thereafter at ten day intervals. Placental infection was detected at 15 days postinfection. Only postinoculation sera collected from postinfected ewes contained antibodies reactive to C. psittaci. Five (26%) chlamydial infected ewes experienced inapparent fetal loss before day 105 of gestation. This finding is significant since C. psittaci infection in sheep is commonly associated with abortion and not infertility.     

Use of the IDEIA ELISA to detect Chlamydia psittaci (ovis) in material from aborted fetal membranes and milk from ewes affected by ovine enzootic abortion

The IDEIA ELISA was used to detect Chlamydia psittaci (ovis) antigen in ewes' milk to which were added serial dilutions of chlamydiae titrated as inclusion forming units (ifus) in McCoy cell tissue culture. The test was able to detect as few as 35 ifus/ml of the organism. The ELISA was then used to detect chlamydial antigen in fetal membranes and milk from ewes clinically affected with ovine enzootic abortion (OEA). The results were compared with results of isolation of chlamydiae in McCoy cell tissue culture from the same material. The fetal membranes of 17 of 19 ewes were positive for chlamydia when tested with the ELISA but chlamydia could be cultured from only 15 of them. Milk samples from 26 ewes which had aborted between 1 and 34 days previously were tested: chlamydiae could not be cultured from any of them and only one was positive when tested by the ELISA. The results show that the IDEIA ELISA is a sensitive test for the detection of C. psittaci (ovis) antigens. The positive results to this test for the three samples from which chlamydiae could not be cultured suggest that the test is not as specific as culture or that it detected dead organisms. Chlamydiae do not appear to be excreted in the milk of ewes affected with OEA.     

Detection of chlamydial antibody by fetal serology--an aid to the diagnosis of ovine abortion.

Two serological tests (indirect immunofluorescence and enzyme-linked immunosorbent assay) were developed for the detection of fetal antibody to Chlamydia psittaci. Fetal blood and thoracic fluid from 126 field cases of suspected ovine chlamydial abortion were examined using both tests. Placenta and fetal tissues (lung, liver, and kidney) from the same animals were also examined by the following conventional diagnostic methods: isolation in McCoy cells, detection of chlamydial lipopolysaccharide (LPS), modified Ziehl-Nielsen staining, and direct fluorescent antibody staining of chlamydia in frozen cryostat sections. Seventy cases were positive by fetal serology, and of these, 68 were also positive by isolation and/or LPS detection. The remaining 56 cases had negative fetal serology, and of these, 39 were positive by isolation and/or LPS detection. Results indicate that fetal serology, although less sensitive than either isolation in McCoy cells or detection of chlamydial LPS antigen, may be of particular use when placenta is not available.     

Comparison of isolates of Chlamydia psittaci of ovine, avian and feline origin by analysis of polypeptide profiles from purified elementary bodies

The single species Chlamydia psittaci is a diverse grouping which contains several different types of chlamydial strain for which there is no generally accepted typing method. The results obtained when profiles of polypeptides from purified elementary bodies are compared are consistent with type designations obtained using other criteria. However, the method still requires large scale culture and extensive purification of the chlamydial cells.     

High prevalence of chlamydial (Chlamydophila psittaci) infection in fetal membranes of aborted equine fetuses

Seventy-seven cases of equine abortion from 49 Hungarian farms that occurred between 1998 and 2000 were investigated for the presence of chlamydiae by immunohistochemistry, PCR and/or MZN staining. Evidence of the presence of these bacteria was obtained in 64 cases (83.1%) from 41 (83.7%) different farms. Partial ompA gene sequencing of PCR products revealed that the agent was Chlamydophila psittaci. Based on the findings of microbial diagnosis, pathology and case history, chlamydial infection was considered to be the most likely cause of abortion in at least 11 (14.3%) cases. In the remaining 53 Chlamydophila-positive cases, either other bacterial and viral agents (n = 22 or 28.6%) as well as non-infectious factors (n = 14 or 18.2%) were identified as more probable primary causes of disease, or the role of chlamydiae remained unclear because lesions in fetuses and fetal membranes were absent (n = 17 or 22.1%). When chlamydial antigen was detected in aborted equine placental tissue using immunohistochemistry it was seen only in the chorionic epithelial cells, but not in other parts of the fetal membranes nor in any of the fetal tissues. In conclusion, chlamydial infection of the genital tract should be considered a possible factor in equine reproductive disorders.     

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)     

Characterisation of Chlamydia psittaci isolated from a horse

This paper describes the isolation and characterisation of a strain of Chlamydia psittaci obtained from a nasal swab taken from a horse with serous nasal discharge. Initial isolation was achieved in cycloheximide-treated McCoy cell monolayers. Chlamydial inclusions stained by immunofluorescence either with a rabbit antiserum raised against C. psittaci or with a monoclonal antibody directed against the genus-specific lipopolysaccharide antigen were single and compact. They did not stain with iodine or with a monoclonal antibody reactive against Chlamydia trachomatis. The agent was re-isolated in the yolk sacs of embryonated hens eggs and designated N16. Identification of the agent was confirmed by electron microscopy. Unique plasmid DNA was prepared from a purified suspension of chlamydial elementary bodies (EBs), and analysed by electrophoresis through 1.0% agarose gels stained by ethidium bromide. This strain of C. psittaci grew relatively slowly in cycloheximide-treated McCoy cells, and the yield of elementary bodies during the course of one growth cycle was relatively low.     

Detection and antigenicity of chlamydial proteins that bind eukaryotic cell membrane proteins.

Chlamydia psittaci proteins capable of binding eukaryotic cell membranes were identified and antigenically characterized. Cell membrane proteins (CMP) of noninfected cells were labeled with biotin (B-CMP), then were extracted with 1% Triton X-100. Nitrocellulose membrane strips containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of chlamydial elementary bodies (EB) were reacted with the B-CMP extract, followed by addition of streptavidin-conjugated horse radish peroxidase. Among the various strains of chlamydiae examined, a protein of approximately 16 to 18 kDa consistently bound B-CMP. A second larger protein, ranging in molecular mass from 24 to 32 kDa, also bound B-CMP. Immunoblotting techniques were used to analyze the reactions of antisera from immunized and experimentally infected animals to these proteins. A rabbit polyclonal antiserum produced against the 18-kDa adhesin of a serovar-1 strain of C psittaci (B577) reacted strongly with 18-kDa proteins of all C psittaci strains, but weakly with that of C trachomatis. Mouse antisera raised against the serovar-2 (FC-Stra) 28-kDa protein reacted only with proteins of the homologous serovar. Sera from experimentally infected animals did not react with the C trachomatis 18-kDa adhesion protein, but did react in 2 patterns with related and nonrelated C psittaci isolates. Two rabbits inoculated with infective serovar-1 EB and 1 rabbit inoculated with a serovar-2 strain reacted specifically with the 18-kDa proteins of their homologous serovars. In contrast, 2 other rabbits inoculated with the same serovar-2 strain produced antisera that reacted with all C psittaci 18-kDa proteins, as did serum from a similarly inoculated bull.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serological response to pgp3 protein in animal and human chlamydial infections

Specific antibodies to plasmid-encoded protein pgp3 are known to be encountered in human Chlamydia (C.) trachomatis infections. In order to verify whether antibodies to this protein could be developed in animals infected with plasmid-carrying chlamydial strains, 454 animal sera were examined using a home-made pgp3 protein ELISA and Western blots (WB) of recombinant pgp3 protein from Chlamydophila (Cp.) psittaci. Likewise, 50 human sera were tested by ELISA and WB of recombinant pgp3 from C. trachomatis. The reactivity against pgp3 protein was compared to the reactivity against chlamydial elementary bodies (EBs) detected by microimmunofluorescence (MIF) test. The presence of pgp3-specific antibodies was demonstrated in most ducks and pigeons with Cp. psittaci infection detected by MIF, as well as in the majority of symptomatic cats and pigs infected with Cp. felis and C. suis, respectively, which reacted at high titres to Cp. felis and C. suis EBs by MIF. Moreover, most of the sera collected from patients with C. trachomatis culture-confirmed infection and seropositive to C. trachomatis by MIF, presented antibodies specific to C. trachomatis pgp3 recombinant protein. Therefore, pgp3 protein could be a useful marker of chlamydial infections in animals, as well as in humans.     

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.     

Conjunctival swab cytology from a guinea pig: it's elementary!

Three 3-month-old guinea pigs (Cavia porcellus) were evaluated for purulent ocular discharge. Conjunctival swabs were obtained for cytologic evaluation of Wright's-Giemsa-stained preparations. The specimen from the most severely affected guinea pig consisted primarily of karyolytic neutrophils and small lymphocytes. Epithelial cells occasionally were observed that contained intracytoplasmic coccoid basophilic organisms, 0.5-1.5 microm in diameter. The intraepithelial inclusions were most consistent with Chlamydia sp elementary and reticulate bodies. Specimens from the other 2 guinea pigs had a similar inflammatory response, but organisms were not observed. Polymerase chain reaction (PCR) analysis of a conjunctival swab from the most severely affected guinea pig was positive for C psittaci, which also is referred to as Chlamydophila caviae, immunotype 8, formerly known as the guinea pig inclusion conjunctivitis strain of C psittaci. Chlamydial conjunctivitis is a common problem in guinea pig populations, with C caviae being specific for this species. Cytologic identification of elementary or reticulate bodies within epithelial cells is diagnostic for the organism in Giemsa-stained preparations. However, PCR is an important complementary tool when organisms are not observed and for accurate classification of the Chlamydia species.     

Seroprevalence of Chlamydia psittaci-specific antibodies in small stock in Namibia--epidemiological study with an enzyme-linked immunosorbent assay (ELISA)

An IgG (H+L)-ELISA was applied as a screening test for antibodies against Chlamydia (C.) psittaci in sera of goats and sheep in Namibia. In 576 (27.3%) of a total of 2,107 sera (299 = 25.2% of 1,185 caprine and 277 = 30.0% of 922 ovine sera) chlamydial antibodies could be detected. 86% of all farms tested revealed seropositive animals. Chlamydial infections were prevalent in all the geographical regions tested. The infection rates per State Veterinary District varied from 12.0% (Otjiwarongo) to 50.0% (Otavi) in goats and from 13.3% (Otjiwarongo) to 41.7% (Windhoek) in sheep. The regional distribution of chlamydial infections was not related to geographical or climatic factors. Sera from herds showing symptoms indicative for chlamydial infections showed significantly higher antibody rates (35% in goats and 39% in sheep) than sera from herds without health problems (18% in goats and 24% in sheep). Considering only sera from farms with clinical history of chlamydiosis, high seroprevalences were correlated to the symptoms abortion and keratoconjunctivitis. As in other countries, enzootic abortion seems to be the main manifestation of chlamydial infection in small ruminants in Namibia. C. psittaci might also play a considerable role in the etiology of infectious keratoconjunctivitis, whereas association with other clinical entities seems to be rare.     

Chlamydial infection in aborted and stillborn lambs

Chlamydia psittaci is a major cause of ovine abortion in the fourth to fifth months of gestation. During the lambing seasons of 1986, 1987, and 1988, fetuses from 52 cases of ovine abortion, stillbirth, or perinatal death were submitted to the laboratory for necropsy examination. Placenta or fetal tissues from 34 cases were cultured on mouse L cells for C. psittaci. Chlamydia psittaci was identified by immunofluorescence on cultures in 20 of these cases. The major gross lesion consistently associated with chlamydial abortion was placentitis with multifocal cotyledonary necrosis and accumulation of red-brown exudate in the intercotyledonary placenta. Chlamydiae appeared as spherical organisms, less than 1 micron in diameter, in the cytoplasm of trophoblasts in impression smears of cotyledons. Histologically, placentitis was sometimes accompanied by pneumonia or encephalitis in the fetus. Chlamydia psittaci was considered the cause for fetal death when chlamydial isolation was associated with placentitis or inflammation of other fetal tissues and when other abortifacient agents were not detected.     

Direct isolation of the agent of enzootic abortion of ewes (Chlamydia psittaci) in cell cultures

Isolation of Chlamydia psittaci in McCoy cell coverslip cultures from clinical material from field cases of enzootic abortion of ewes proved more sensitive than diagnosis by examination of smears stained by Ziehl-Neelsen. Enzootic abortion could be diagnosed in the absence of fetal membranes by culture of fetal lung or liver tissue.     

鹦鹉热嗜性衣原体感染SPF鸡和污染相关疫苗的初步调查

为初步调查SPF鸡感染鹦鹉热嗜性衣原体状况及相关SPF鸡胚源疫苗是否出现污染,本试验通过采集不同日龄的SPF鸡血清70份、SPF种蛋卵黄膜30份,收集市场上销售的SPF鸡胚源疫苗共41支,利用国产间接血凝试剂盒检测抗体,进口免疫荧光试剂盒分别测定其抗体、抗原阳性率,以评价SPF鸡鹦鹉热嗜性衣原体的流行状况和相关疫苗的污染状况。本试验结果显示,SPF鸡血清阳性率分别为31.4%(荧光法)、5.7%(间接血凝法);SPF种蛋阳性率33.3%,SPF鸡胚源疫苗平均阳性率31.7%。SPF鸡已经感染了鹦鹉热嗜性衣原体,且发现经鸡胚卵黄膜而传播病原的新途径,进而造成SPF鸡胚源疫苗出现衣原体污染。因此,加强SPF鸡鹦鹉热嗜性衣原体监测已势在必行。     

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.