Evaluation of two commercial assays for the detection of Chlamydophila abortus antibodies.

Two commercial enzyme-linked immunosorbent assays (ELISA), the CHEKIT-CHLAMYDIA which uses inactivated Chlamydophila psittaci antigen, and the Chlamydophila abortus ELISA produced by the Institut Pourquier which uses a recombinant fragment of the 80-90 kDa protein, were evaluated with the objective to determine whether the new ELISAs would perform as improved alternatives to the complement fixation test (CFT) for the serological diagnosis of ovine enzootic abortion (OEA). The results were compared to those obtained by the CFT and the competitive ELISA (cELISA). The tests were assessed with a panel of 17 serum samples from specific pathogen-free (SPF) lambs experimentally infected with various subtypes of Chlamydophila pecorum, with sera from 45 C. abortus-infected pregnant sheep and from 54 sheep free of OEA. The C. abortus ELISA was identified as being more specific and sensitive than the other tests. The 4 assays were evaluated further with 254 sera from flocks with documented OEA, from flocks with no history of abortion and from animals after abortion of unknown cause. The C. abortus ELISA by the Institut Pourquier identified less OEA-positive sera than the other assays though it identified correctly 9 of 10 OEA-positive flocks. The basis of the discordant results is discussed.

Lymphatic regeneration following hind limb replantation: an experimental study in the dog.

Different types of trauma to the lymphatic system can often occur, but surgical intervention can be performed only in specific cases. We report on lymphatic regeneration following limb replantation in traumatic amputations and replantation of extremities. The aim of this study was to observe the progression and reaction after surgical trauma that is similar to other kinds of trauma, both in children or adults, and to monitor the possible lymphatic regeneration. Particular attention was paid to two parameters: firstly, the physical examination of the replanted limbs by checking the post-traumatic lymphoedema, and secondly, the study of the images taken from indirect lymphangiography of the replanted limbs. Histological specimens of the surgical trauma area were also examined to reconfirm or exclude lymphatic regeneration. The study population consisted of sixteen mongrel dogs, divided into two groups of eight animals each, who underwent hind limb elective amputation and replantation combined with (group A) or without (group B) sciatic nerve division. Lymphoedema formation was followed quantitatively by measurement of the circumference of the hind limb for 21 days after replantation. Indirect lymphography, never performed before in such cases, and histopathology, were performed to evaluate and confirm lymphatic regeneration. Lymphatic regeneration after replantation of the operated hind limbs was first confirmed between 7th and 11th postoperative day by indirect lymphangiography and clinical observation of the post-traumatic lymphoedema of these limbs. The mean time of visualisation of lymphatic regeneration through lymphography was 10.12 days for group A and 9.37 days for group B. However, nerve transection had no effect on lymphatic regeneration (p = 0.46). Histopathological examination showed first evidence of lymphatic regeneration on the ninth postoperative day and a network of newly formed capillary lymphatics on the 21st postoperative day. It is concluded that lymphatic regeneration following replantation of the extremities without anastomosing of the interrupted lymph vessels, is an unquestionable fact. To achieve the best lymphatic drainage and use of the replanted extremities it is important to resect all non-vital tissues of the replantation area. Local or general infections decelerate lymphatic regeneration. Indirect lymphography with iotrolan is a reliable, easy to perform technique without complications that may be used repeatedly for confirmation and evaluation of post-traumatic lymphoedema.

Seroprevalences for ovine enzootic abortion in Switzerland.

Our aim was to assess the seroprevalence of Chlamydophila (Cd) abortus (Chlamydia psittaci serotype 1), denoted ovine enzootic abortion (OEA), in the Swiss sheep population. A competitive enzyme-linked immunosorbent assay (cELISA) was adapted for the investigation of pooled serum samples (pool approach) and receiver-operator characteristic (ROC) analysis was applied to define the cut-off of the pool approach. At a cut-off value of 30% inhibition, the flock-level pooled sensitivity and specificity were 92.9% and 97.6% when compared to classifying the flock based on individual-animal samples. Subsequently, sera from 775 randomly selected flocks out of 11 cantons of Switzerland were investigated using the pool approach. The cantons included in the study represented 72% of the Swiss sheep flocks and 76% of Swiss sheep population. Antibodies against Cd. abortus were found in almost 19% (144) of the 775 examined sheep flocks. Test prevalences were adjusted for the imperfect test characteristics using the Rogan-Gladen estimator and Bayesian inference. Seroprevalence was highest (43%) in the canton Graubunden. In the remaining 10 cantons the seroprevalence ranged from 2 to 29%. The cELISA in combination with testing pooled sera and statistical methods for true prevalence estimation provided a good survey tool at lower costs and time when compared to other approaches.

[Ovine enzootic abortion: seroprevalence in Switzerland using a competitive enzyme-linked immunosorbent assay (cELISA)]. / Chlamydienabort beim Schaf: Untersuchung der Seroprävalenz in der Schweiz mittels eines kompetitiven ELISA (cELISA).

The present study gives an overview over the seroprevalence of ovine enzootic abortion in Switzerland. 639 sheep flocks out of eight cantons in Switzerland were examined by a competitive enzyme linked immunosorbent assay (cELISA) for antibodies against Chlamydophila abortus (Chlamydia psittaci serotype 1), the agent causing ovine enzootic abortion. The eight cantons included Aargau, Bern, Zürich, Appenzell-Ausserrhoden, Appenzell-Innerrhoden and Fribourg, the Vallais and the Graubünden. They were representative for 57% of the Swiss sheep flocks and for 60% of Swiss sheep population. In total, almost 19% (118) of the examined flocks were seropositive. Seroprevalence was the highest in Graubünden with 41%; this requires further examination and the evaluation of the need for a monitoring and controlling program. The examination of pooled sera made it possible to test a large number of samples with a reasonable amount of work. Higher sensitivity (92.9%) and specificity (97.6%) than the complement fixation test (CFT) in combination with testing of pooled sera makes the cELISA to be an usuable tool for serological screening on flock level.

Polymorphic outer-membrane proteins of Chlamydophila abortus are glycosylated.

Antigenic profiles of mono-, bi- and poly-specific monoclonal antibodies against 90 kDa polymorphic outer-membrane proteins (POMPs) and a 105 kDa POMP-related protein of Chlamydophila abortus ATCC VR 656(T), after one- and two-dimensional electrophoretic analysis, helped identify each one of the triplets POMP 90, 91A and 91B, and a POMP-related protein at 85 kDa. The lectin concanavalin A bound to the four POMPs and the POMP-related protein in a specific manner and the binding was sensitive to treatment with the amidase N-endoglycosidase F, suggesting the presence of small asparagine-linked oligosaccharide chains. The exposure of the five proteins on the chlamydial surface and the orientation of the attached oligosaccharide chains was examined by protease and endoglycosidase treatments of intact bacteria. The results were consistent with the concept that some of the oligosaccharides in the POMPs face outwards, possibly protecting the polypeptides from proteolytic enzymes, whereas the oligosaccharides in the 105 kDa POMP-related protein are oriented inwards, thereby rendering the polypeptide chain accessible to proteases. A possible role for the N-linked oligosaccharides in the POMPs might be the promotion of the proper folding and processing of these proteins.

Experimental infection of pregnant ewes with enteric and abortion-source Chlamydophila abortus.

Two groups of pregnant ewes were experimentally infected oronasally in midpregnancy. A faecal and an abortion-source isolate of Chlamydophila abortus were used. They were derived from a healthy ewe from a flock with no history of abortion, and from an aborted foetus in a farm with enzootic abortion. As assessed by modified Ziehl-Neelsen (MZN) staining, egg culture, antigen ELISA, the Clearview test and immunohistochemistry, inoculation resulted in placental and/or foetal infection in all ewes. Histopathology revealed placentitis in two and four ewes inoculated with the enteric or abortion-source isolate, respectively, in addition, these samples were immunohistochemically positive for chlamydial antigen. All six ewes infected with the enteric isolate and five of seven ewes infected with the abortion-source isolate showed evidence for a serological response by an indirect ELISA or CFT. Neither chlamydiae nor lesions were detected in the placentae and lambs of the uninfected control ewes, which remained seronegative. Our results suggest that enteric C. abortus can be associated with placental and foetal lesions in sheep.

Diagnosis of ovine enzootic abortion using an indirect ELISA (rOMP91B iELISA) based on a recombinant protein fragment of the polymorphic outer membrane protein POMP91B of Chlamydophila abortus.

Chlamydophila abortus is of major economic importance worldwide as one of the principal causes of abortion in sheep. Serological diagnosis of infection by the complement fixation test (CFT) is complicated by false positive reactions resulting from cross-reactive antibodies to Chlamydophila pecorum. To improve diagnosis an indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant protein fragment of the C. abortus polymorphic outer membrane protein POMP91B (rOMP91B iELISA) was assessed using a panel of 281 sera from experimentally and naturally infected sheep. The iELISA performed well, being more sensitive (84.2%) and specific (98.5%) than the CFT. Furthermore, the iELISA was better at differentiating C. abortus- from C. pecorum-infected animals. The new rOMP91B iELISA test will prove a valuable tool for the routine serodiagnosis of C. abortus infection.

Identification of protective epitopes by sequencing of the major outer membrane protein gene of a variant strain of Chlamydia psittaci serotype 1 (Chlamydophila abortus).

Protective monoclonal antibodies (MAbs) to the major outer membrane protein (MOMP) of species of the family Chlamydiaceae, which is the primary vaccine candidate antigen, recognize nonlinear epitopes conferred by the oligomeric conformation of the molecule. Protective MAbs failed to recognize oligomeric MOMP of the variant strain LLG, which bears amino acid substitutions in variable segments (VSs) 1, 2, and 4, and competed with monomer-specific MAbs mapping to these VSs in reference strain 577. The results suggest that multiple sites located in the three VSs contribute to the epitope of protective MAbs.

Molecular characterization of a bacteriophage (Chp2) from Chlamydia psittaci.

Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C. psittaci RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the phiX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein.

[Chlamydia abortion in sheep: possibilities for serological diagnosis using a competitive ELISA and insight into the epidemiologic situation in Switzerland]. / Chlamydienabort beim Schaf: Möglichkeiten der serologischen Diagnostik mit einem kompetitiven ELISA und Einblick in die epidemiologische Situation in der Schweiz.

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.

T2 relaxation time study of iron overload in b-thalassemia.

Myocardial iron deposition occurs as a result of blood transfusion therapy in b-thalassemia major patients. Since this deposition causes various cardiac complications, it is of interest to assess the iron content of the myocardium in relation to the clinical picture of the patients. Two different MRI indices were used to achieve this purpose: the T2 relaxation time and the heart/skeletal muscle signal intensity ratio. ECG gated spin echo images were obtained from 54 adult thalassemic patients, with a mean age of 26 (18-44) years, at TE = 22 ms and 60 ms, using a 1.5 T system. Patients were divided into 2 groups (A and B), according to their serum ferritin levels (> or < 2000 ng ml(-1)). Results were compared with nine controls, with a mean age of 25 (18-43) years. Heart T2 relaxation time in controls (44.3 +/- 3.5 ms) was higher than in group A (29.9 +/- 5.7 ms, P < 0.001) and group B (33.4 +/- 6.8 ms, P < 0.01). T2 was measurable in 66% of group A and 83% of group B patients. The heart/muscle signal intensity ratio in group A (0.45 +/- 0.27) was lower than in group B (0.82 +/- 0.33, P < 0.001) and the controls (1.15 +/- 0.20, P < 0.001). The heart/muscle signal intensity ratio was measurable in 94% of the patients and demonstrated an inverse relationship with the serum ferritin levels (r = -0.52, P < 0.01). This study indicates that the heart/muscle ratio is a sensitive index of iron overload and it can be measured in the majority of patients, irrespective of tissue iron concentration, thereby offering an advantage over the use of T2 relaxation time.

Immunoelectron microscopic localisation of the OMP90 family on the outer membrane surface of Chlamydia psittaci.

The putative outer membrane location of the OMP90 (formerly POMP) family from the ovine abortion strain of Chlamydia psittaci was investigated by immunoelectron microscopy. Using a non-embedding technique, antigens were shown to be localised on the outer membrane surface of both elementary and reticulate bodies, the infectious and non-infectious forms of Chlamydiae respectively. Antibodies affinity-purified against the expressed amino- and carboxy-terminal halves of one of the family members. OMP90A, demonstrated that the amino half is surface-exposed while the carboxyl half is most probably localised internally. Surface localisation on elementary bodies indicates the importance of these proteins as protective antigen candidates.

Diagnosis of ovine enzootic abortion, using a competitive ELISA based on monoclonal antibodies against variable segments 1 and 2 of the major outer membrane protein of Chlamydia psittaci serotype 1.

OBJECTIVE: To develop and evaluate a competitive ELISA (cELISA) specific for detection of antibodies to abortion strains of Chlamydia psittaci and C pecorum that is based on monoclonal antibodies against the 2 segments. PROCEDURA: Monoclonal antibodies were screened for binding to ELISA plates coated with elementary bodies, and were selected on the basis of positive competition with experimentally produced sera against C psittaci and lack of competition with anti-C pecorum sera. The cELISA was evaluated with field sera, and the results were compared with those obtained by complement-fixation testing and by an ELISA containing solubilized outer membrane complexes (A-ELISA). RESULTS: The cELISA detected 9 of 10 C psittaci-infected flocks (57/125 sera, 45.6%), and in 6 of 10 flocks (27.3% of the sera), it specified correctly the infecting chlamydial species. Regarding test sensitivity, the complement-fixation test detected 6 of 10 test-positive (19.2% of the sera) flocks, whereas 7 of 10 test-positive (48.8% of the sera) flocks were detected by use of the A-ELISA. The specificity of the test was satisfactory (100%), compared with the A-ELISA (72.2%). CONCLUSIONS: The new cELISA is a sensitive and specific assay for antibodies against C psittaci abortion strains. It is rapid and easy to perform and does not require serum dilutions. The new cELISA is, therefore, suitable as a routine test for chlamydial diagnosis and seroepidemiologic studies.

Increased type IV collagen-degrading activity in metastases originating from primary tumors of the human colon.

The secretion of specific matrix metalloproteinases (MMPs) is considered a prerequisite step for tumor cell invasion and metastasis. In the present study we investigated the expression of type IV collagen-degrading activity in primary tumors of the human colon in correlation to tumor grade and in comparison to activity expressed in arising metastases. We observed that type IV collagen-degrading activity (MMP-2 and MMP-9), purified by ion exchange, gel filtration and affinity chromatography and characterized by gelatin zymography, correlates to tumor grade. Furthermore, in surgical specimens identified as metastases originating from primary tumors of the colon, we observed that enzyme activity was significantly enhanced, relatively to that identified in the primary tumor. This observation should be considered when targeting MMPs as a therapeutic intervention to prevent cancer progression.

Two-dimensional electrophoretic analysis of the protein family at 90 kDa of abortifacient Chlamydia psittaci.

Four major clusters, designated A, B, C and D, were distinguished in Western Blots by a monoclonal antibody specific for the "antigen family at 90 kDa" after two-dimensional electrophoretic analysis on immobilized pH gradient of chlamydial elementary bodies of abortifacient C. psittaci. Clusters B, C, and D were closely related with molecular mass (kDa) pI values of 91.5/5.2-5.4, 90/5.0-5.2 and 90.5/5.6-5.8, respectively. Cluster A was larger, with molecular mass/pI of 104.7/5.1-5.3. Evidence for the antigenic relationship between cluster A and clusters B, C and D was further supported by immunological cross-reaction with affinity-purified antibodies from serum of ewes with chlamydial-induced abortion. The experimental values obtained for size and pI of the four clusters correlated well with the calculated values from known sequences coded by multiple chlamydial genes. Direct evidence for the correspondence between the immunoreactive clusters B, C and D and the retrieved genes was provided by antibody binding experiments to recombinant polypeptides representing fragments of the deduced proteins. The 4-member antigen family at 90/104 kDa is the first example of proteins coded by multiple genes within the genus Chlamydia.

Diversity among abortion strains of Chlamydia psittaci demonstrated by inclusion morphology, polypeptide profiles and monoclonal antibodies.

Twenty eight C. psittaci abortion strains had been previously classified in to 4 immunologically distinct groups on the basis of cross-protection experiments in a mouse model. To identify the molecular basis of their immunological divergence 4 representative strains were investigated by cellular, molecular and immunological techniques. An identical pattern was obtained by Alul digestion of the amplified major outer membrane protein gene (MOMP) by the polymerase chain reaction (PCR) of the 4 strains. However, inclusion morphology and polypeptide profiles clearly distinguished one strain, named LLG, and its homologous strain POS from the other prototypes by the presence of a unique protein at 26.5 kDa and the absence of a polypeptide at 23 kDa. Six out of 10 monoclonal antibodies (mAbs) raised against abortion strains failed to react with inclusions of the 2 strains. All 6 mAbs reacted with the chlamydial outer membrane complex (COMC). Two of these mAbs, one against the MOMP and one against an antigen at 90 kDa, did not react with immunoblots of LLG and POS. The data provide direct demonstration of the existence of strain variation in the field and classify strains LLG and POS as a distinct C. psittaci serotype 1-subtype. The antigenic diversity among abortion strains should be taken into consideration when designing a subunit vaccine.

Mapping of Chlamydia trachomatis proteins by immobiline-polyacrylamide two-dimensional electrophoresis: spot identification by N-terminal sequencing and immunoblotting.

Proteins from purified elementary bodies of Chlamydia trachomatis were separated by two-dimensional gel electrophoresis on nonlinear wide-range immobilized pH gradients in the first dimension and polyacrylamide gradient gels in the second dimension. The maps obtained with this system are highly reproducible and resolve ca. 600 spots. By using immunoblot analysis with specific antibodies and/or N-terminal amino acid sequencing, we established the map positions of a number of described chlamydial proteins, such as the major outer membrane protein (MOMP) the 60 kDa cystein-rich outer membrane protein (OMP2), the DnaK-like, GroEL-like, and macrophage infectivity potentiator (MIP)-like proteins, the plasmid-encoded pgp3 protein, two ribosomal proteins (S1 and L7/L12), and the protein-elongation factor EF-Tu. Other proteins, for which gene assignment was not possible, have been identified by three parameters (Mr, pI and N-terminal sequence). This work provides a preliminary basis for a future and progressive compilation of a genome-linked database of chlamydial proteins.

Heterogeneity within the first constant segment of the major outer membrane protein gene in Chlamydia trachomatis serovar D/Da distinguishes 2 lineages.

Restriction fragment length polymorphism of the major outer membrane protein gene (ompl) was studied in 73 epidemiologically unrelated Chlamydia trachomatis serovar D (n = 64) and Da (n = 9) strains isolated between 1983 and 1991 in various european countries from patients suffering from sexually transmitted diseases, as well as 3 reference strains D/IC-Cal-8 (USA), D/UW3 (UK) and Da/TW-448 (Taiwan). Among these strains, 5 different genotypic groups were distinguished: 3 for the serovar D strains and 2 for the serovar Da strains. Comparisons of the complete ompl nucleotide sequence of 1 member of each group revealed 2 distinct lineages, independently of the D/Da classification, presumably as the result of an intragenic DNA recombination event within the first constant segment. The observed ompl genetic diversity of serovar D/Da strains in regions involved in T- and B-cell responses might add another level of complexity to the design of a subunit vaccine.

The role of lipopolysaccharide in the exposure of protective antigenic sites on the major outer membrane protein of Chlamydia trachomatis.

A species-specific monoclonal IgM antibody (mAb) 9BF8 directed against the major outer membrane protein (MOMP) of Chlamydia trachomatis neutralized several chlamydial serovars in a complement-independent manner. The presence of Mg2+ ions negated the neutralization in serovars F, L1 and L2, but not in serovars A, B, E, D and K. The ability of monovalent Fab-fragments of this mAb to neutralize chlamydial infectivity in a Mg-independent manner suggested that conformational alterations on the chlamydial surface induced by the cation hindered the IgM but allowed the smaller Fab fragment access to its epitope. In order to determine the chlamydial component that binds Mg, elementary bodies (EB) of serovars E and L1 were treated with EDTA at pHs 8 and 9. The infectivity of the treated EB and the amount of released LPS were determined. Only after EDTA treatment at pH 9, as the LPS release increased, did the binding of the mAb on the chlamydial surface become Mg-independent. The infectivity of the EB was almost completely lost after such a treatment. These results suggest that the chlamydial LPS has the potential to modulate the exposure of antigenic sites on the MOMP, when it is cross-linked by Mg2+. They further imply that serovars protected by Mg and those that are not differ in the surface topology of one particular MOMP epitope, but are antigenically very similar. This difference might be of considerable importance in vivo.