Growth and development of tetracycline-resistant Chlamydia suis.

Tetracycline (TET) is a front-line antibiotic for the treatment of chlamydial infections in both humans and animals, and the emergence of TET-resistant (Tet(r)) Chlamydia is of significant clinical importance. Recently, several Tet(r) chlamydial strains have been isolated from swine (Sus scrofa) raised in production facilities in Nebraska. Here, the intracellular development of two Tet(r) strains, R19 and R27, is characterized through the use of tissue culture and immunofluorescence. The strains grow in concentrations of up to 4 microg of TET/ml, while a TET-sensitive (Tet(s)) swine strain (S45) and a strain of the human serovar L2 (LGV-434) grow in up to 0.1 microg of TET/ml. Although inclusions form in the presence of TET, many contain large aberrant reticulate bodies (RBs) that do not differentiate into infectious elementary bodies. The percentage of inclusions containing typical developmental forms decreases with increasing TET concentrations, and at 3 microg of TET/ml 100% of inclusions contain aberrant RBs. However, upon removal of TET the aberrant RBs revert to typical RBs, and a productive developmental cycle ensues. In addition, inclusions were found that contained both C. suis R19 and Chlamydia trachomatis L2 after sequential infection, demonstrating that two biologically distinct chlamydial strains could both develop within a single inclusion.

Intramolecular secondary structure rearrangement by the kissing interaction of the Neurospora VS ribozyme.

Kissing interactions in RNA are formed when bases between two hairpin loops pair. Intra- and intermolecular kissing interactions are important in forming the tertiary or quaternary structure of many RNAs. Self-cleavage of the wild-type Varkud satellite (VS) ribozyme requires a kissing interaction between the hairpin loops of stem-loops I and V. In addition, self-cleavage requires a rearrangement of several base pairs at the base of stem I. We show that the kissing interaction is necessary for the secondary structure rearrangement of wild-type stem-loop I. Surprisingly, isolated stem-loop V in the absence of the rest of the ribozyme is sufficient to rearrange the secondary structure of isolated stem-loop I. In contrast to kissing interactions in other RNAs that are either confined to the loops or culminate in an extended intermolecular duplex, the VS kissing interaction causes changes in intramolecular base pairs within the target stem-loop.

Avian chlamydiosis.

Avian chlamydiosis (AC) can be economically devastating to producers and a serious public health problem. Most infections in humans are due to exposure to psittacine birds and pigeons; however, outbreaks resulting in severe disease and even death do occur in abattoir workers following processing of infected flocks. The disease occurs primarily in turkeys and ducks, but can affect all types of poultry. In poultry, the disease varies from one producing high morbidity and mortality to one that is asymptomatic. Farm workers and abattoir workers are at risk following exposure to either extreme. Although outbreaks of AC have declined since the 1970s, some parts of the world are now experiencing a rise in incidence. Whether the initial decrease was due to changes in production methods or to the increased use of antibiotics is not known. The mechanism for introduction of the disease into a flock or area is poorly understood. Wild birds are often infected by the same strains as domestic flocks and are therefore thought to play a major role in introduction. Data also indicate that vertical transmission may occur. Persistently infected carrier birds are known to be a source of chlamydiosis in the pet bird industry, but have not been confirmed as a source of infection in poultry flocks.

Rearrangement of a stable RNA secondary structure during VS ribozyme catalysis.

The Neurospora VS ribozyme recognizes and cleaves a substrate RNA that contains a GC-rich stem loop. In contrast to most RNA secondary structures that are stable during tertiary or quaternary folding, this substrate undergoes extensive ribozyme-induced rearrangement in the presence of magnesium in which the base pairings of at least seven of the ten nucleotides in the stem are changed. This conformational switch is essential for catalytic activity with the wild-type substrate and creates a metal-binding secondary structure motif near the cleavage site. Base pair rearrangement is accompanied by bulging a cytosine from the middle of the stem, indicating that ribozymes may perform base flipping, an activity previously observed only with protein enzymes that modify DNA.

Intestinal lesions caused by a strain of Chlamydia suis in weanling pigs infected at 21 days of age.

The objective of this study was to determine whether a strain of Chlamydia suis shown previously to be an intestinal pathogen in gnotobiotic piglets could cause diarrhea and intestinal lesions in young weanling pigs. Pigs from 2 sows were randomly assigned to 2 groups. Group 1 included 13 pigs that were weaned at 24 hours of age and then housed in isolator units and fed milk replacer and unmedicated starter ration. Group 2 included 8 pigs that nursed their respective sows, consumed unmedicated starter ration, and were weaned at 21 days of age. Ten pigs in group 1 and 6 pigs in group 2 were inoculated orally with 4 x 108 inclusion-forming units of C. suis strain R27 at 21 days of age. Control pigs were inoculated with sham inoculum. The pigs were necropsied 5-14 days postinoculation (DPI). None of the Chlamydia-infected pigs developed diarrhea. Villus atrophy was seen histologically in sections of ileum from Chlamydia-infected pigs in both groups 5 and 7 days DPI. Lymphangitis and multiple lymphohistiocytic and neutrophilic aggregates were seen in the submucosa, tunica muscularis, and serosa of the distal jejunum, ileum, and colon from Chlamydia-infected pigs in both groups 5-14 DPI. Immunostaining of sections of distal jejunum, ileum, and colon from infected pigs revealed chlamydial antigen in intestinal epithelium and in foci of lymphangitis/inflammation. The results indicated that C. suis strain R27 can cause intestinal lesions in young weanling pigs, and the lesions are similar to those seen in gnotobiotic piglets. The results also indicated that strain R27 causes asymptomatic intestinal infections in young weanling pigs, at least under the conditions of this study.

Conjunctivitis caused by a swine Chlamydia trachomatis-like organism in gnotobiotic pigs.

The objective of this study was to determine whether a chlamydial strain recovered from growing and finishing swine with conjunctivitis or keratoconjunctivitis could cause the same infections in gnotobiotic pigs. The strain shares biological characteristics with Chlamydia trachomatis. After propagation in Vero cells and preparation of the inoculum (10(7) inclusion-forming units/ml), chlamydial strain H7 was instilled into the ventral conjunctival sac (0.15 ml/sac) of 12 anesthetized 3-day-old gnotobiotic piglets. Four age-matched gnotobiotic piglets were anesthetized and sham infected with uninfected cell culture lysates. None of the principal piglets developed clinical symptoms of conjunctivitis or keratoconjunctivitis. Principal piglets necropsied 7 days postinfection (DPI) had histologic lesions of mild or moderate conjunctivitis; immunohistochemical evaluation revealed chlamydial antigen in conjunctival epithelium. A majority of principal piglets necropsied at 14-28 DPI had histologic lesions of mild conjunctivitis, but chlamydial antigen was not detected by immunohistochemistry. The results indicated that chlamydial strain H7 can cause mild or occasionally moderate conjunctivitis in gnotobiotic pigs, but the conjunctival infection is asymptomatic.

Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms.

The current taxonomic classification of Chlamydia is based on limited phenotypic, morphologic and genetic criteria. This classification does not take into account recent analysis of the ribosomal operon or recently identified obligately intracellular organisms that have a chlamydia-like developmental cycle of replication. Neither does it provide a systematic rationale for identifying new strains. In this study, phylogenetic analyses of the 16S and 23S rRNA genes are presented with corroborating genetic and phenotypic information to show that the order Chlamydiales contains at least four distinct groups at the family level and that within the Chlamydiaceae are two distinct lineages which branch into nine separate clusters. In this report a reclassification of the order Chlamydiales and its current taxa is proposed. This proposal retains currently known strains with > 90% 16S rRNA identity in the family Chlamydiaceae and separates other chlamydia-like organisms that have 80-90% 16S rRNA relatedness to the Chlamydiaceae into new families. Chlamydiae that were previously described as 'Candidatus Parachlamydia acanthamoebae' Amann, Springer, Schönhuber, Ludwig, Schmid, Müller and Michel 1997, become members of Parachlamydiaceae fam. nov., Parachlamydia acanthamoebae gen. nov., sp. now. 'Simkania' strain Z becomes the founding member of Simkaniaceae fam. nov., Simkania negevensis gen. nov., sp. nov. The fourth group, which includes strain WSU 86-1044, was left unnamed. The Chlamydiaceae, which currently has only the genus Chlamydia, is divided into two genera, Chlamydia and Chlamydophila gen. nov. Two new species, Chlamydia muridarum sp. nov. and Chlamydia suis sp. nov., join Chlamydia trachomatis in the emended genus Chlamydia. Chlamydophila gen. nov. assimilates the current species, Chlamydia pecorum, Chlamydia pneumoniae and Chlamydia psittaci, to form Chlamydophila pecorum comb. nov., Chlamydophila pneumoniae comb. nov. and Chlamydophila psittaci comb. nov. Three new Chlamydophila species are derived from Chlamydia psittaci: Chlamydophila abortus gen. nov., sp. nov., Chlamydophila caviae gen. nov., sp. nov. and Chlamydophila felis gen. nov., sp. nov. Emended descriptions for the order Chlamydiales and for the family Chlamydiaceae are provided. These families, genera and species are readily distinguished by analysis of signature sequences in the 16S and 23S ribosomal genes.

Identification of nine species of the Chlamydiaceae using PCR-RFLP.

The family Chlamydiaceae contains two genera and nine species. Rapid and easy identification of these species is essential for taxonomic, epidemiological and clinical determinations. Currently, DNA sequence analysis is the only accepted method that decisively distinguishes all nine species. In this study, a simple and rapid PCR-RFLP procedure was developed by which laboratory-cultured chlamydial specimens could be identified. To accomplish this, conserved oligonucleotide primers and restriction sites were deduced from 16S and 23S rRNA sequence data from > 50 chlamydial strains representing all nine species. DNA from 25 previously characterized chlamydial strains were tested with these primers and restriction enzymes. All nine chlamydial species were reliably distinguished in the tests. The procedure was optimized by adjusting the annealing temperature using both a standard and a heat-activated DNA polymerase to reduce mismatch PCR amplification of mycoplasmas and other bacteria. The result was that a PCR method for species identification of chlamydial isolates and for distinguishing mycoplasmas and chlamydiae was created. This method can be used to rapidly identify known species of the family Chlamydiaceae.

Serotyping of chlamydial clinical isolates from birds with monoclonal antibodies.

Forty-nine avian chlamydial strains, isolated mainly from various regions in France and from different species of birds, were analyzed and tested with a panel of nine monoclonal antibodies (MAbs) by the indirect microimmunofluorescence test (MIF). The MAbs included five serovar-specific MAbs, three MAbs raised against Chlamydia psittaci and Chlamydia pecorum ovine strains, and one genus-specific MAb. Of the 49 isolates, 41 came from parrots or budgerigars; the rest were from pigeons, a canary, a duck, and a dove. Two additional strains were from unknown hosts. Most of these avian strains were successfully serotyped according to their reactions with five serovar-specific MAbs by the MIF test. The serovars of 44 strains were determined: 39 were of serovar A, 3 of serovar B, and 2 of serovar E. The remaining five isolates were unclassified because they did not react with any of five serovar-specific MAbs but did react with genus MAb or the MAbs produced with ovine strains. The five unclassified isolates (two from budgerigars, two from Gabon gray parrots, and one from a duck) indicate that one or more additional serovars of C. psittaci exist in birds. The heterogeneity within each subgroup was evident because the 49 avian isolates gave 10 subgroups when the results of the five serovar-specific MAbs were combined with results from the three MAbs produced with ovine strains. This heterogeneity of the serovar isolates, as shown by the combination of MAbs, could provide strain markers very useful for epidemiologic studies.

In situ hybridization for the detection and localization of swine Chlamydia trachomatis.

Gnotobiotic piglets were inoculated intralaryngeally with swine Chlamydia trachomatis strain R33 or orally with swine C. trachmatis strain R27. Archived formalin-fixed, paraffin-embedded tissues from piglets euthanatized 4-7 days postinoculation were examined by in situ hybridization for C. trachomatis nucleic acid using a nonradioactive digoxigenin-labeled DNA probes that targeted specific ribosomal RNA or omp1 mRNA molecules of the swine C. trachomatis strains. Positive hybridization signals were detected in bronchial epithelial cells, bronchiolar epithelial cells, pneumocytes, alveolar and interstitial macrophages, and jejunal and ileal enterocytes. Chlamydia-infected cells had a strong signal that was confined to the intracytoplasmic inclusions. Positive hybridization signals were not detected in tissue sections from an uninfected control piglet or in C. psittaci-infected sheep placenta. The morphology of host cells was preserved despite the relatively high temperature required in parts of the incubation procedure. The data indicate that in situ hybridization can be used to detect swine C. trachomatis in formalin-fixed, paraffin-embedded tissue specimens.

Rapid detection of the Chlamydiaceae and other families in the order Chlamydiales: three PCR tests.

Few identification methods will rapidly or specifically detect all bacteria in the order Chlamydiales, family Chlamydiaceae. In this study, three PCR tests based on sequence data from over 48 chlamydial strains were developed for identification of these bacteria. Two tests exclusively recognized the Chlamydiaceae: a multiplex test targeting the ompA gene and the rRNA intergenic spacer and a TaqMan test targeting the 23S ribosomal DNA. The multiplex test was able to detect as few as 200 inclusion-forming units (IFU), while the TaqMan test could detect 2 IFU. The amplicons produced in these tests ranged from 132 to 320 bp in length. The third test, targeting the 23S rRNA gene, produced a 600-bp amplicon from strains belonging to several families in the order Chlamydiales. Direct sequence analysis of this amplicon has facilitated the identification of new chlamydial strains. These three tests permit ready identification of chlamydiae for diagnostic and epidemiologic study. The specificity of these tests indicates that they might also be used to identify chlamydiae without culture or isolation.

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.

Two new serovars of Chlamydia psittaci from North American birds.

Five Chlamydia psittaci isolates (1 turkey, 1 psittacine, 1 human, and 2 pigeon isolates) failed to react with serovar-specific monoclonal antibodies to known avian and mammalian C. psittaci serovars and were presumed to represent 1 or more new serovars. The isolates were characterized using restriction endonuclease analysis of the whole genome, polymerase chain reaction-restriction fragment length polymorphism of the major outer membrane protein genome, monoclonal antibody comparisons, and growth in tissue culture. Monoclonal antibodies were produced to the human isolate (MP) and to the psittacine isolate (VS225). The monoclonal antibody results show that the isolates represent 2 new avian serovars (serovars E and F). The restriction fragment length polymorphism analysis of the major outer membrane protein genome demonstrated that the isolates are distinct. The whole genome restriction endonuclease analysis data and the growth patterns in tissue culture indicate that the new serovars are similar to avian serovars recognized previously. A subspecies monoclonal antibody that reacted with serovars A and B also reacted with serovar E, indicating that these serovars are closely related. The results show that these isolates represent 2 new avian serovars, making them the fifth and sixth avian serovars identified in North American birds.

Chlamydiosis in captive white-winged doves (Zenaida asiatica).

Chlamydia psittaci was isolated from the spleen of a moribund white-winged dove (Zenaida asiatica). The isolate was serotyped as the serovar B that is commonly isolated from pigeons. A fourfold increase in the titer of antichlamydial IgM activity occurred in that bird in paired serum samples tested by chlamydial elementary body agglutination (EBA) and a greater than or equal to fourfold decrease of IgG occurred by direct complement fixation (DCF). The increases or decreases of EBA and DCF titers in other clinically ill birds that were treated with tetracycline varied, as normally occurs in cases of avian chlamydiosis. Titers in clinically normal birds were consistent with past infections. These birds were from a captive group of about 200 birds to be used for breeding and reproduction research. A small sample of recently caught wild birds was serologically negative for chlamydial antibody activity.

The ribosomal intergenic spacer and domain I of the 23S rRNA gene are phylogenetic markers for Chlamydia spp.

Current methods used to classify Chlamydia strains, including biological, morphological, and DNA hybridization techniques and major outer membrane protein (omp1) gene analysis, can be imprecise or difficult to perform. To facilitate classification, 2.8-kb partial ribosomal DNA (rDNA) segments from a Chlamydia trachomatis strain and a Chlamydia psittaci strain were amplified by PCR and sequenced. Subsequently, a 1,320-bp region in this segment, including both the 16S/23S intergenic spacer (232 +/- 11 bp) and domain I (620 +/- 2 bp) of the 23S gene, was sequenced from 41 additional strains and from the chlamydia-like organisms Simkania sp. strains "Z" and "Z1." When both parsimony and distance analyses were performed, these sequences were found to have variable regions that grouped the isolates into two lineages (C. trachomatis and non-C. trachomatis) and nine distinct genotypic groups. The C. trachomatis lineage included human, swine, and mousehamster groups. The non-C. trachomatis lineage included Chlamydia pecorum, Chlamydia pneumoniae, and C. psittaci abortion, avian, feline, and guinea pig groups. These nine groups were essentially equidistant from the genetic root and were congruent with groups identified previously by using DNA-DNA homology, genomic restriction endonuclease analysis, host specificity, tissue specificity, and/or disease production. Phylogenetic trees based on the intergenic spacer or on domain I were congruent with trees previously derived from ompI sequences. DNA sequence analysis of either the intergenic spacer or domain I provides a rapid and reproducible method for identifying, grouping, and classifying chlamydial strains.

Intestinal lesions caused by two swine chlamydial isolates in gnotobiotic pigs.

The objective of this study was to determine whether 2 distinct chlamydial isolates recovered from the intestines and feces of diarrheic nursery pigs could cause intestinal lesions in gnotobiotic pigs. Both isolates share biological characteristics with Chlamydia trachomatis. Chlamydial isolates R27 and R19 were propagated in Vero cells or embryonated eggs, respectively, and suspended in sucrose-phosphate-glutamine buffer with 10% fetal bovine serum for inoculation. Sham inocula were prepared from uninfected cell culture lysates and from uninfected eggs. Each piglet was fed 1 ml of inoculum or sham inoculum at 3-4 days of age. Ten piglets were each fed 10(9) inclusion-forming-units (IFU) and 14 piglets were each fed 10(6) IFU of isolate R27; 5 control piglets were fed sham inoculum. Twenty piglets were each fed 10(5) IFU R19; 5 control piglets were fed sham inoculum. All infected piglets developed diarrhea 4-5 days postinfection (DPI). Most piglets fed 10(9) IFU R27 became anorexic, dehydrated, and weak and were necropsied 4-7 DPI. Piglets fed 10(6) IFU R27 or 10(5) IFU R19 were necropsied 4, 7, 10, 14, and 18 DPI. Diarrhea, although never profuse, persisted in the piglets fed 10(6) IFU R27 or 10(5) IFU R19 through 12 DPI. At necropsy, all diarrheic piglets had watery colonic contents with flecks of undigested curd. In small intestine, histologic lesions were seen most consistently in distal jejunum and ileum. Distal jejunum and ileum from piglets fed 10(9) IFU R27 and necropsied 4-5 DPI were characterized by villus atrophy and multifocal necrosis of villi; necrosis was limited to the tips or apical one half of villi. Mild to severe villus atrophy, lymphangitis, and perilymphangitis were seen in the distal jejunum and ileum from all infected piglets 7 and 10 DPI. Colon from 1 infected piglet necropsied 10 DPI had mild focal serositis; significant colonic lesions were not seen in the other infected piglets. Immunostaining done on sections of distal jejunum and ileum revealed chlamydial antigen in villus enterocytes, occasional goblet cells, and occasional cryptal enterocytes and in foci of lymphangitis and perilymphangitis; the amount of detectable chlamydial antigen decreased after 4 DPI. In colon, sparse positive staining was seen in surface enterocytes and cryptal enterocytes. Ultrastructural examination of ileal villus enterocytes revealed chlamydiae, often together with glycogen particles, in vacuoles or occasionally free in the cytoplasm. The results indicated that the swine chlamydial isolates used in this study are intestinal pathogens in gnotobiotic pigs.

Comparison of pharyngeal, fecal, and cloacal samples for the isolation of Chlamydia psittaci from experimentally infected cockatiels and turkeys.

Direct comparisons were made of chlamydial isolation rates from pharyngeal swabs, fecal samples, and cloacal swabs from cockatiels and pharyngeal and cloacal swabs from turkeys experimentally infected with Chlamydia psittaci. During pathogenesis studies, 133 paired specimens were collected from cockatiels and 118 paired specimens were collected from turkeys. Of the 51 cockatiel chlamydial infections detected, 80.4% were positive by the pharyngeal swab sample, 45.1% were positive by the fecal swab sample, and 37.3% were positive by the cloacal swab sample. Of the 87 turkey infections detected, 93.1% were positive by the pharyngeal swab sample and 77.0% were positive by the cloacal swab. The pharyngeal swabs were the most reliable sample for isolation of chlamydia from live birds. However, no single sampling site yielded positive results from all infected birds. Specimens from multiple sites are recommended because a number of infected birds were identified by isolation from only 1 sample.

Serotype 2-specific antigens from ruminant strains of Chlamydia pecorum detected by monoclonal antibodies.

A panel of 25 monoclonal antibodies (MAbs) selected from seven different cellular fusions was used to study the antigenic relationships among a group of 18 ruminant serotype 2 strains of Chlamydia pecorum by indirect microimmunofluorescence test. The antigenic relationships between the strains of C. psittaci serotype 1 and strains of serotype 2 C. pecorum were also studied, as well as the C. pecorum strains and an avian and a feline strain of C. psittaci. Only the genus-specific MAb, used as a positive control, reacted with all the tested strains. Six MAbs reacted with all the C. pecorum serotype 2 strains, but only one of them recognized an 80 kDa protein in Western blot. With these 18 serotype 2 strains, 16 different patterns were established, underlying the high heterogeneity of this group. Only the three caprine strains exhibited the same profile. None of the MAbs reacted with the serotype I strains, or the feline isolate of C. psittaci, but two of them recognized the avian strain. We discuss the possibility that the serotype 2-specific antigens represent C. pecorum species-specific antigens that could be used for diagnosis.

Lung and nasal lesions caused by a swine chlamydial isolate in gnotobiotic pigs.

The objective of this study was to determine whether a chlamydial isolate recovered from nasal swabs from swine with pneumonia could cause pneumonia and rhinitis in gnotobiotic pigs. The identity of the isolate currently is unknown, but it shares characteristics with Chlamydia trachomatis. After propagation in Vero cells and preparation of the inoculum (2.5 x 10(10) inclusion-forming units/ml), chlamydiae were instilled into nostrils (1.0 ml/nostril) and lungs (2.0 ml intralaryngeally) of 15 anesthetized 3-day-old gnotobiotic piglets. Five age-matched gnotobiotic piglets were anesthetized and sham infected with uninfected cell culture lysates. Two treated piglets were moribund and 2 were severely dyspneic prior to necropsy 7 days postinfection (DPI), whereas remaining treated piglets showed mild dyspnea upon exertion throughout the study. All treated piglets developed diarrhea. All treated piglets necropsied 7-21 DPI had extensive consolidation in cranial, middle, and accessory lung lobes; a majority of these piglets also had extensive consolidation in the caudal lobes. Treated piglets necropsied 28 and 35 DPI had a lobular pattern of consolidation in all lung lobes. Histologically, lesions in lungs from treated piglets necropsied 7, 14, and 21 DPI were characterized by bronchointerstitial pneumonia with foci of type II pneumocyte hypertrophy and hyperplasia; pneumocytes and bronchial and bronchiolar epithelial cells were markedly vacuolated. Alveolar macrophages, peribronchitis, peribronchiolitis, and perivasculitis were seen in lungs from treated piglets necropsied 28 and 35 DPI; those necropsied 28 DPI also had foci of lymphohistiocytic and plasmacytic infiltrates. Turbinate lesions in all treated piglets were characterized by mild multifocal lymphoplasmacytic and occasionally neutrophilic rhinitis. Immunohistochemistry detected chlamydial antigen in bronchial and bronchiolar epithelial cells, pneumocytes, and inflammatory cells in treated piglets necropsied 7, 14, and 21 DPI. Positive staining was limited to alveolar macrophages in treated piglets necropsied 28 and 35 DPI. Chlamydial antigen was detected in turbinate epithelial cells at all necropsy intervals. Ultrastructurally, chlamydiae were seen with glycogen particles in vacuoles or free in the cytoplasm of bronchial and bronchiolar epithelial cells and pneumocytes. The results indicated that the chlamydial isolate used in this study is a pathogen in gnotobiotic pigs.