Chlamydiales in guinea-pigs and their zoonotic potential

The aim was to detect and characterize chlamydial infections in guinea-pigs (GP) with ocular disease, study their pathogenicity and zoonotic potential and to test for the presence of Acanthamoebae spp. in GP eyes and to investigate whether they could act as vectors for Chlamydia-like organisms. Overall 126 GP, of which 77 were symptomatic, were screened by clinical examination, cytology, gross pathology, histology, immunohistochemistry, polymerase chain reaction (PCR) and bacteriology. A new Chlamydiaceae-specific intergenic spacer rRNA gene PCR, designed to amplify this segment linking the 16S and 23S regions, was performed. DNA samples were also received from one owner including samples of his cat and rabbit. Guinea-pigs: 48 of 75 symptomatic, but only 11 of 48 asymptomatic GP were positive by PCR for Chlamydophila caviae guinea-pig inclusion conjunctivitis (GPIC) (P < 0.0001). Eighteen of 75 or 15/48, respectively, were positive for DNA from Chlamydia-like organisms. Acanthamoebae-DNA could be found in two GP, of which one was symptomatic. Owner, cat and rabbit: Samples of all three species were positive by PCR for C. caviae GPIC and the owner's one-day disposable contact lenses showed a positive PCR result for the Chlamydia-like organism Parachlamydia acanthamoebae. No Acanthamoebae-DNA could be detected. This study is the first to describe Chlamydia-like organisms in GP and to detect C. caviae GPIC in human, cat and rabbit. Therefore, C. caviae GPIC could pose a zoonotic potential. We believe that the finding of C. caviae GPIC in species other than GP is probably not unique.     

Detection of chlamydiae in boar semen and genital tracts

Chlamydiae cause abortion and reproductive disorders in sows. Although organisms can infect the male genital tract, little is known about the disease situation in boars. Hence, we examined the prevalence of chlamydial infection in semen and genital tracts of boars. Samples collected from Swiss boars (group A: n=42), and boars from Germany (group B: n=39) were examined by bacteriology, LPS-ELISA, immunohistochemistry (IHC) and polymerase chain reaction (PCR). The latter methodology involved use of three PCR assays including 16Sig rDNA, IGS-S (intergenic spacer 16S/23S-Short) and IGS-L (intergenic spacer 16S/23S-Long) PCR for comparison methods. PCR sensitivity and the presence of potential PCR inhibitors were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. Detection limits of the 16Sig and IGS-S PCR were 10 templates, while the IGS-L PCR was less sensitive (100 templates). Of 25 semen samples that were collected from group A, one semen sample was positive for Cp. psittaci and two were positive for Chlamydia-like organisms by 16Sig PCR. Screening of sera from Swiss boars revealed three animals with positive reactions in the LPS-ELISA, although we failed to detect chlamydiae within organs of these or sera-negative animals by IHC or IGS-S PCR. In group B, 10 ejaculates were positive for Chlamydia (C.) suis and two were positive for Chlamydia-like organisms by 16S PCR. The identification of DNA from Chlamydia-like organisms in semen from both groups of boars was surprising and a role for these bacteria in reproductive diseases requires further assessment. In conclusion, the prevalence of chlamydial infection was low in group A animals indicating that venereal transmission may not be significant for Chlamydia-associated reproductive diseases in pigs, although rare cases may occur.     

Evidence of infection in tortoises by Chlamydia-like organisms that are genetically distinct from known Chlamydiaceae species

Nasal lavage fluid was collected from 155 tortoises, mostly Testudo spp., that were kept as companion animals and suffered from nasal discharge. Examination for chlamydial DNA by PCR assays targeting the ompA, ompB, and groESL genes, as well as the 16S rRNA signature region and the 16S-23S intergenic spacer, respectively, revealed 16 (10.3%) positive animals. Sequence analysis of PCR products indicated high homology to the family Chlamydiaceae. Phylogenetic trees constructed from partial sequences of the ompA and 16S rRNA genes showed that the present samples clustered outside the nine species of Chlamydia and Chlamydophila. Sequences of the nearest relative, Chlamydophila pecorum, were still clearly distinct from those of the positive tortoise samples. This suggests that the tortoises had been infected by Chlamydia-like agents, the taxonomic identity and pathogenic importance of which has yet to be established.     

Chlamydiae in free-ranging and captive frogs in Switzerland

A total of 210 frog samples originating either from a mass mortality (1991/1992) or from routine postmortem investigations of the years 1990 to 2004 were examined retrospectively for a possible involvement of Chlamydiae. For a prevalence study of Chlamydia in a selected Swiss amphibian population, 403 samples from free-ranging Rana temporaria were examined. Histopathology, immunohistochemistry using a monoclonal antibody against chlamydial lipopolysaccharide, and a 16S rRNA polymerase chain reaction (PCR) followed by DNA sequencing were performed on the formalin-fixed and paraffin-embedded tissues. Using PCR, 8 of 54 (14.8%) frog samples from the mass mortality (1991/1992) were positive for Chlamydia suis S45. A control group of healthy Xenopus laevis had 3 of 38 positive samples, sequenced as C suis S45 (2/3) and an endosymbiont of Acanthamoeba species UWE1 (1/3). Chlamydophila pneumoniae TW-183 was detected from exotic frogs kept in a zoo. Of the frogs collected for the prevalence study, 6 of 238 (2.5%) tested positive, 1 each for C suis S45, Cp pneumoniae TW-183, and uncultured Chlamydiales CRG22, and the remaining 3 revealed Chlamydophila abortus S26/3. In immunohistochemistry, there were 2 positive labeling reactions, 1 in intestine and the other in the epithelium coating the body cavity, both testing positive for Cp pneumoniae TW-183 in PCR. Histologically there were no lesions recorded being characteristic for Chlamydia. Although there is a prevalence of Chlamydia in Swiss frogs, no connection to a mass mortality (1991/1992) could be established. For the first time, C suis S45 and Cp abortus S26/3 were detected in frog material.     

Respiratory chlamydial infection based on experimental aerosol challenge of pigs with Chlamydia suis

An experimental study of aerogeneous challenge in pigs was conducted in order to reveal characteristic features of porcine respiratory chlamydiosis. Eight conventionally raised pigs were exposed to a pathogenic strain of Chlamydia (C.) suis, four controls were mock infected. Besides pathological changes, the acute-phase and humoral immune responses, as well as the dissemination and transmission of the challenge strain was monitored in the course of infection. The data from clinical investigations, LPS-binding protein assay, antibody ELISAs, confocal laser scanning and light microscopy, immunohistochemical staining and PCR provided extensive evidence of the pathogenic potential of C. suis for the porcine respiratory system. This model appears suitable for further pathophysiological and immunological investigations of chlamydial respiratory infections and can also be recommended for studies of Chlamydia-associated infections of the human lung.     

Molecular evidence for chlamydial infections in the eyes of sheep

Ocular infections by chlamydiae are associated with ocular disease manifestations such as conjunctivitis and keratitis in humans and animals. Limited evidence exists that members of the order Chlamydiales can also cause ocular disease in sheep. In the current study, the prevalence of chlamydiae in the eyes of sheep was investigated by using PCR methods. Data obtained in sheep by broad-range 16S rRNA order Chlamydiales-specific PCR were compared to the prevalence of antibodies against chlamydiae detected by a competitive enzyme-linked immunosorbent assay (cELISA). Flocks tested included a clinically healthy flock and two flocks suffering from ocular disease and with histories of Ovine Enzootic Abortion (OEA). PCR detected DNA of Chlamydophila (Cp.) abortus and Cp. pecorum in the eyes of both healthy and sick animals but also identified Chlamydia (C.) suis and a variety of uncultured chlamydia-like organisms. Good correlation was found between the presence of Cp. abortus DNA in sheep conjunctival samples and seropositivity detected by cELISA. Despite these findings, no association was found between the presence of chlamydial DNA in the sheep conjunctival samples and the onset of clinical disease. These results suggest that the biodiversity of chlamydiae in the eyes of sheep is greater than that previously thought. Further investigations are needed to determine whether a causal relationship between infection by chlamydiae and ocular disease exists in these animals.     

Survey of intestinal parasites in pigs from intensive farms in Guangdong Province, People's Republic of China

The prevalence of intestinal parasites was investigated in intensive pig farms in Guangdong Province, China between July 2000 and July 2002. Faecal samples from 3636 pigs (both sexes and five age groups) from 38 representative intensive pig farms employing different parasite control strategies were examined for the presence of helminth ova and protozoan oocysts, cysts and/or trophozoites using standard techniques. Of the 3636 pigs sampled, 209 (5.7%) were infected with Trichuris suis, 189 (5.2%) with Ascaris, 91 (2.5%) with Oesophagostomum spp., 905 (24.9%) with coccidia (Eimeria spp. and/or Isospora suis) and 1716 (47.2%) with Balantidium coli. These infected pigs were mainly from farms without a strategic anti-parasite treatment regime. Concurrent infection of multiple parasites was common, and T. suis was the most common nematode infecting breeding, young and mature pigs. The results of the present investigation provide relevant 'base-line' data for assessing the effectiveness of control strategies against intestinal parasitism in intensively raised pigs in Guangdong Province, China.     

An experimentally induced Chlamydia suis infection in pigs results in severe lung function disorders and pulmonary inflammation

This study was aimed at evaluating the pathophysiology of pulmonary dysfunctions and inflammatory consequences of an acute respiratory chlamydial infection induced experimentally in conventionally raised pigs (aged 39-44 days). Eight animals were exposed to Chlamydia suis (C. suis) and four non-infected animals served as controls. The total observation period was from seven days before challenge to seven days post exposure. While non-infected control pigs did not exhibit any clinical symptoms, animals exposed to C. suis developed fever and were severely respiratory distressed within the first week after exposure. After C. suis infection, pulmonary dysfunctions were characterised by a significant decrease in the diffusion capacity of the lung (i.e. transfer factor of the lung for carbon monoxide; TL CO), a significant increase in the functional residual capacity (FRC), and significant changes in the pattern of ventilation (respiratory rate increased while the tidal volume decreased). In exhaled breath condensate (EBC), leukotriene B(4) (LTB(4)) and interleukin 6 (IL-6) showed a tendency to increase after infection. In the broncho-alveolar lavage fluid (BALF) of C. suis infected pigs, the activity of matrix metalloprotease 9 (MMP-9) was found to be increased compared to controls. BALF cytology was characterised by increased numbers of granulocytes and activated lymphocytes. Pulmonary inflammation in infected pigs was confirmed by post mortem histology. A prominent dissemination of chlamydial bodies in the lung was accompanied by an influx of macrophages, granulocytes and activated T-cells. Data obtained in this study provide new insight into the pathogenesis of acute respiratory chlamydial infections in pigs.     

Evaluation of lung function in pigs either experimentally or naturally infected with Chlamydiaceae

This study evaluated the influence of chlamydial infections on lung function in conventionally raised pigs. Eight pigs aged 39-44 days were included in an aerogeneous challenge model (4 were exposed to Chlamydia suis; 4 served as controls). Nineteen pigs aged 5-27 weeks without clinical symptoms (but partly PCR-positive for chlamydial species) were examined over 6 months. For lung function testing, impulse oscillometry was used. In total, all 27 pigs underwent 465 lung function tests. Variables of ventilation (respiratory rate, tidal volume, minute volume), respiratory impedance (expressed as resistance and reactance within the frequency range 3-15 Hz), and model derived resistance of proximal and distal airways were measured. Experimental exposure to C. suis significantly affected lung function. Early distal airway obstruction (3-5 days after infection) was followed by an obstruction of proximal airways (7 days after infection). The breathing pattern was significantly changed (decreased tidal volume; increased respiratory rate). In symptom-free pigs having a naturally acquired presence of different chlamydial species in the respiratory system, no deterioration in lung function could be determined up to the age of 6 months. In conclusion, the consequences of respiratory chlamydial infections appear to vary from clinical inapparence to severe respiratory distress.     

Waddlia, Parachlamydia and Chlamydiaceae in bovine abortion

The etiology remains unknown in many cases of bovine abortion in Switzerland. Bacteria of the Chlamydiales order are known abortive agents, therefore cases of bovine abortion from three representative regions of Switzerland were investigated in this study. Particularly Chlamydiaceae as well as the Chlamydia-like organisms Waddlia and Parachlamydia were of interest, especially because of their possible zoonotic potential. Placenta samples (n=343) were tested for these bacteria by different PCR-methods, immunohistochemistry and serology for Chlamydia abortus. Additionally an attempt for the isolation of Waddlia and Parachlamydia was made by co-cultivation in amoebae. In 67.3% of the 343 cases a necrotizing and/or purulent placentitis was found histologically. By real-time PCR, 0.9% (3/343) of the cases were positive for Waddlia, 13.4% (46/343) positive for Parachlamydia and 14.6% (50/343) positive or questionable positive for Chlamydiaceae. Of these samples, confirmation by immunohistochemistry was possible in 2/3 cases for Waddlia, 25/46 for Parachlamydia and 4/50 for Chlamydiaceae. Of the 50 cases positive or questionable positive for Chlamydiaceae, species-identification by ArrayTube Microarray or 16S rRNA PCR resulted in 41 cases positive for C. abortus whereas the presence of Chlamydia suis was confirmed in four and Chlamydia pecorum in one case. This study brought evidence for the importance of different members of Chlamydiales in different regions of Switzerland although Waddlia is not occurring in a high prevalence. On the other hand mixed infections with different Chlamydiales as well as with other abortigenic agents could be found.     

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.     

Aberrant chlamydial developmental forms in the gastrointestinal tract of pigs spontaneously and experimentally infected with Chlamydia suis

The phenomenon of persistence is well known from in vitro studies, where it is associated with the production of aberrant bodies, but its occurrence in vivo is less well documented. The objective of this study was to search for aberrant bodies in intestinal tissues from pigs, describe their ultrastructure, and investigate the suitability of immunohistochemical staining for chlamydial heat shock protein 60 (cHSP60) to detect such forms. Intestinal tissues derived from pigs naturally and experimentally infected with Chlamydia (C.) suis were examined by immunohistochemistry, transmission electron microscopy and immunogold electron microscopy. The chlamydial species involved in the natural infection were determined using an Array Tube Microarray to C. suis and Chlamydophila abortus. Ultrastructurally, aberrant bodies were detected in the gut of both naturally and experimentally infected pigs. Immunogold electron microscopy showed that the aberrant bodies were labeled less strongly than the normal forms by antibodies against LPS and cHSP60 respectively. It was concluded that aberrant bodies occur in vivo in pigs and that the gnotobiotic pig model might be suitable for the study of chlamydial persistence in vivo. The antibody against cHSP60 does not appear to be suitable to specifically detect such forms.     

猪附红细胞体16S rRNA基因的序列测定和系统进化分析

从确诊为猪附红细胞体感染的猪场，无菌采集血样，抽提猪附红细胞体基因组DNA，采用真细菌的通用引物进行16S rRNA基因扩增，对扩增产物进行克隆和测序。从3个地理位置不同的猪场均成功地扩增出长度为1469bp的核苷酸序列。系统进化分析表明，3个猪场样品所测序列一致性达99．52％以上，具有相同的基因型，但与国外报道的猪附红细胞体Illinois株同源性为95％，属于同一基因群，但基因型不同；所有种类的附红细胞体和血巴尔通氏体组成同一进化分支，这类血营养菌与支原体科，支原体属的病原最靠近(75％)，而与立克次氏体目的病原较远(70％)。上述研究证实，广东所流行的猪附红细胞体是一种新基因型的猪附红细胞体，建议命名为猪附红细胞体广东株型；为反映进化关系，猪附红细胞体和其它血营养菌应划归于支原体科的支原体属。     

Experimental enteric infection of gnotobiotic piglets with Chlamydia suis strain S45

Enteric chlamydial infections of pigs with Chlamydia (C.) suis are frequent and often subclinical. The enteric pathogenicity of C. suis strain S45 was investigated in gnotobiotic piglets. Piglets from three litters (n=31) were inoculated with egg-grown chlamydiae at 2-3 days of age (n=17) or used as controls (n=14). They were observed for clinical signs, killed and necropsied sequentially at 2-13 days postinoculation (DPI). Feces were collected daily and investigated with an ELISA for chlamydial antigen. At necropsy, specimens were collected for histopathology and for immunohistochemical, PCR-based, and serological (complement fixation test, ELISA) detection of chlamydiae. Chlamydial replication and associated symptoms and lesions were observed from 2 to 13 DPI and were particularly pronounced within the first week PI. Clinical symptoms consisted of moderate-to-severe diarrhea, slight and transient anorexia, weakness and body weight loss. Immunohistochemistry and ELISA revealed that chlamydial replication was particularly marked at 2-4 DPI and primarily located in the small intestinal villus enterocytes. Further sites of replication included large intestinal enterocytes, the lamina propria and Tunica submucosa, and the mesenteric lymphnodes. Histopathological changes included moderate-to-severe villus atrophy with flattened enterocytes and focal villus tip erosions, and moderate mucosal inflammatory cell infiltrates and lymphangitis in the small intestine. PCR of spleen tissue and blood was mostly negative for chlamydiae, indicating that they did not substantially disseminate into the host up to 13 DPI. All sera were negative for anti-chlamydial antibodies. In conclusion, C. suis strain S45 elicited significant enteric disease and lesions in gnotobiotic piglets indicating its pathogenic potential for swine.     

Selection for tetracycline-resistant Chlamydia suis in treated pigs

The aim of this study was to investigate Chlamydia suis in a pig farm with an outbreak of conjunctivitis and diarrhea. Eye swabs and pooled fecal samples were investigated for the presence of C. suis by real-time PCR and ArrayTube microarray. Samples positive for C. suis by ArrayTube microarray assay were further tested for the presence of the tet(C) resistance gene by PCR. In the first examination, C. suis was identified in 12 six-week-old pigs showing conjunctivitis. Of these, the tet(C) gene-coding region was amplified in one pooled fecal sample and one eye swab, respectively. After oral treatment with tetracycline, clinical symptoms disappeared. Subsequently, all eye swabs investigated from 10 healthy pigs were positive for C. suis and the tet(C) gene-coding region. The present study reports rapid selection for tetracycline-resistant C. suis after antibiotic treatment.     

Diagnostic investigation into the role of Chlamydiae in cases of increased rates of return to oestrus in pigs

Cervical swabs and serum samples were taken from Swiss herds of sows with high rates of irregular return to oestrus (group A) and from control herds without reproductive problems (group B. The genital tracts of 21 slaughtered sows of group A were also examined. The swabs and genital tracts were screened for Chlamydiae by a new 16S rRNA PCR and the sera by an ELISA for Chlamydiaceae lipopolysaccharide. Chlamydophila (Cp) abortus was isolated from seven of the 65 swabs taken from group A but from none of the 128 swabs taken from group B. Chlamydia suis was present in swabs from both groups A (1.5 per cent) and B (2.3 per cent). In addition, Cp abortus was detected in 33.3 per cent of the genital tracts. Of the 193 sera tested, 61.7 per cent were positive, with no significant difference between group A (52.3 per cent) and group B (66.4 per cent). Chlamydia-like organisms were detected in 28.2 per cent of the swabs from group A and in 22 per cent of those from group B.     

Comparison of diagnostic methods to detect piroplasms in asymptomatic cattle

This study was carried out to compare different diagnostic techniques to reveal the presence of piroplasms in asymptomatic cattle kept at pasture. Nineteen blood samples were collected from animals of two different areas of Emilia Romagna Region of Italy and processed for microscopic observation, PCR, serological test (IFAT) for Babesia bovis and Babesia bigemina antibodies and in vitro cultivation. The cultures were performed on both bovine and ovine erythrocytes. Seventeen blood smears (89%) were positive for piroplasms, while PCR was positive on 18 samples (95%). DNA sequencing of 18S rRNA identified the piroplasms as Theileria spp. In vitro cultures were successful for 6 samples (32%) cultured on bovine blood and subsequent identified these as Babesia major by PCR. On IFAT analyses of 16 samples, 36.8% resulted positive for B. bovis and 31.6% positive for B. bigemina. These results show, in the same animals, the co-infection with Babesia spp. and Theileria spp.; the detection of B. major was possible only using the in vitro cultures.     

Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera

Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.     

Feline chlamydiosis

Chlamydiae are an important cause of acute and chronic conjunctivitis in cats. Until recently, only one organism was thought to infect cats, Chlamydophila felis (previously Chlamydia psittaci var. felis). Recently, other Chlamydia-like organisms belonging to the family Parachlamydiaceae, which comprises organisms that reside and proliferate within free-living amoeba, have been identified in cats with neutrophilic and eosinophilic conjunctivitis. The relative importance of these organisms and their amoebic hosts requires investigation. There is also weak evidence that chlamydiae may also be capable of causing reproductive tract disease and lameness in cats. Diagnosis of chlamydial conjunctivitis requires use of specialized culture techniques or the polymerase chain reaction. The antibiotic of choice to treat these infections is doxycycline; azithromycin is less effective. All cats in the household should be treated simultaneously. The zoonotic potential of these organisms appears low, but some precaution is warranted when handling affected cats.     

Detection of novel chlamydiae in cats with ocular disease

OBJECTIVE: To detect and characterize the full range of chlamydial infections in cats with ocular disease by use of polymerase chain reaction (PCR) assays, cytologic examination, immunohistochemical analysis, and evaluation of clinical information including status for feline herpesvirus-1 (FeHV-1). SAMPLE POPULATION: DNA extracted from 226 conjunctival samples obtained from cats with clinically diagnosed keratitis or conjunctivitis and 30 conjunctival samples from healthy cats. PROCEDURE: PCR assays for the 16S rRNA gene specific for the order Chlamydiales and a new Chlamydophila felis (formerly Chlamydia psittaci) species-specific 23S rRNA gene were performed. Seventy-four conjunctival samples were prepared with Romanowsky-type stain, grouped on the basis of inflammatory pattern, and screened for chlamydial inclusions by use of immunohistochemical analysis. Clinical information and FeHV-1 status were recorded. RESULTS: 26 (12%) specimens had positive results for the only known feline chlamydial pathogen, C felis. Surprisingly, an additional 88 (39%) were positive for non-C felis chlamydial DNA. Identification of non-C felis chlamydial DNA by direct sequencing revealed 16S rRNA gene sequences that were 99% homologous to the sequence for Neochlamydia hartmannellae, an amebic endosymbiont. Chlamydial prevalence was significantly higher in cats with ocular disease. CONCLUSIONS AND CLINICAL RELEVANCE: Application of a broad-range detection method resulted in identification of a new agent associated with ocular disease in cats. Finding chlamydia-like agents such as N hartmannellae in coinfections with their obligate amebic host, Hartmannella vermiformis, raises questions about the potential role of these microorganisms in causation or exacerbation of ocular disease in cats.