The application of metagenomic next-generation sequencing in diagnosing Chlamydia psittaci pneumonia: a report of five cases.

BACKGROUND: Chlamydia psittaci pneumonia is a zoonotic infectious disease caused by Chlamydia psittaci. Diagnostic tools, including culture, serologic test and PCR-based methods, are available but prone to false negative results. CASE PRESENTATION: This report included five cases of Chlamydia psittaci pneumonia. Symptoms and signs common to all 5 cases included fever, coughing, generalized muscle ache, and most notably, inflammatory infiltration of the lungs upon chest CT and X-ray. Metagenomic next-generation sequencing (mNGS) revealed the presence of Chlamydia psittaci in biopsy lung tissue in 3 cases and bronchoalveolar lavage fluid in the remaining 2 cases. Three patients responded to doxycycline plus moxifloxacin; two patients responded to moxifloxacin alone. CONCLUSIONS: mNGS could be used to diagnose Chlamydia psittaci pneumonia.

A parrot-type Chlamydia psittaci strain is in association with egg production drop in laying ducks.

Chlamydophila psittaci (C. psittaci) is an avian pathogen associated with systemic wasting disease in birds, as well as a public health risk. Although duck-related cases of psittacosis have been reported, the pathogenicity and shedding status of C. psittaci in ducks are unclear. In this study, we reported that C. psittaci (genotype A) is responsible for a disease outbreak characterized by poor laying performance and severe lesions in multiple organs of ducks. Oral administration of antibiotic, doxycycline, was found to effectively control the C. psittaci infection in laying ducks. Collectively, our new findings provide evidence that C. psittaci was the major pathogen responsible for the outbreak of this disease in ducks. In order to reduce economic losses incurred by this disease, effective control measures must be taken to prevent infection in laying duck farms.

[Problems in the diagnosis of ornithosis].

The paper describes 3 cases of ornithosis that could be detected by clinical and laboratory studies using immunological assays. In one case, its diagnosis was made late when the infection occurred in a woman working in a travel agency in Cyprus. The two other cases having an occupational contact (pet shop workers) were observed to have its acute form. All the described cases showed lung involvement characterized by external respiratory failure, one of the most common manifestations of ornithosis (psittacosis). Among the practically used laboratory tests, the indirect hemagglutination reaction is an accessible and effective serological assay for the diagnosis of ornithosis.

Differential cytokine expression in Chlamydophila psittaci genotype A-, B- or D-infected chicken macrophages after exposure to Escherichia coli O2:K1 LPS.

Chlamydophila (Cp.) psittaci and avian pathogenic Escherichia (E.) coli infections contribute to the respiratory disease complex observed in turkeys. Secondary infection with E. coli exacerbates Cp. psittaci pathogenicity and augments E. coli excretion. The innate immune response initiated by both pathogens in their avian host is unknown. We therefore determined the cytokine responses following Cp. psittaci infection and E. coli superinfection of avian monocytes/macrophages by examining gene transcripts of IL-1beta, IL-6, CXCLi2 (IL-8), CXCLi1 (K60), IL-10, IL-12alpha/beta, IL-18, TGF-beta4 and CCLi2 at 4h post-inoculation with different Cp. psittaci strains or 4h post-treatment with avian E. coli LPS of Cp. psittaci pre-infected HD11 cells. Cp. psittaci strains used were 84/55 and 92/1293 (highly virulent), CP3 (low virulent) and 84/2334 (phylogenetically intermediate between Cp. psittaci and Chlamydophila abortus). At 4h post chlamydial infection, an increased expression of IL-1beta and IL-6 as well as CXCLi2, CXCLi1 and CCLi2 was observed compared to levels in uninfected HD11 controls. This effect was less pronounced for the milder CP3 strain. The pro-inflammatory response of Cp. psittaci infected cells to E. coli LPS was significantly lowered compared to uninfected controls, especially when the cells were pre-infected with highly virulent Cp. psittaci strains. In both experiments, exceptionally high IL-10 and no TGF-beta4 responses were observed, and we propose that this could induce macrophage deactivation and NF-kappaB suppression. Consequently, pro-inflammatory and Th1-promoting responses to both the primary Cp. psittaci infection and E. coli would be inhibited, thus explaining the observed aggravated in vivo pathology.

Experimental Chlamydia psittaci serotype 1 enteric infection in gnotobiotic piglets: histopathological, immunohistochemical and microbiological findings.

The enteric pathogenicity of the ovine C. psittaci serotype 1 isolate S26/3 was assessed using a litter of gnotobiotic piglets. In one group, eight piglets were inoculated at 3 days of age; at 10 days, two of these were re-inoculated. In a second group, six animals were mock-inoculated at 3 days of age as negative controls; subsequently, at 10 days, three of these piglets were inoculated with C. psittaci. The animals were observed for clinical signs, killed and necropsied sequentially between 4 and 17 days of age. At necropsy, specimens were collected for histopathology, immunohistochemistry and serology. Clinical manifestations consisted of sporadic slight softening of faeces observed between 8 and 12 days post inoculation (d.p.i.) in pigs inoculated at 3 days of age and between 4 and 6 d.p.i. in those inoculated at day 10. Histopathological changes were minimal and inconsistent and occurred almost exclusively in the small intestine in pigs of 15 days of age and older; they consisted of a slight shortening of villi, of a small number of tongue-shaped villi and of villous fusions. Immunohistochemistry revealed small numbers of chlamydial inclusions in the small intestinal enterocytes of only five pigs, all killed within 5 d.p.i. An ELISA run on faecal samples collected daily after inoculation from six of the pigs showed that chlamydial antigen was excreted in the faeces. In pigs inoculated at 3 days, chlamydial antigen was detected inconsistently before, and consistently after 9 d.p.i. Pigs inoculated at 10 days excreted antigen consistently after inoculation until the end of their observation period (8 d.p.i.). Infective chlamydiae were detected from the faeces of inoculated piglets using Vero cell cultures. Sera of all pigs were negative for anti-chlamydial antibodies using a complement fixation test. In conclusion, enteric pathogenicity of C. psittaci serotype 1 in a litter of gnotobiotic piglets proved minimal. The results, therefore, indicate that serotype 1 C. psittaci is not likely to cause enteric disease in conventionally reared pigs. Nevertheless, a potential role of swine in the epidemiology of this agent should be considered with regard to spread of Chlamydia to other species.

The production of EAE-free lambs from infected dams using multiple ovulation and embryo transfer.

This investigation aimed to ascertain whether embryo transfer was a feasible method of breaking the disease cycle caused by Chlamydia psittaci (ovis). Ten naive ewe lambs were inoculated orally with the T76 and G188 isolates of C. psittaci (ovis) in late pregnancy. Five animals which sero-converted to the complement fixation test (CFT) were used as donors for a multiple ovulation and embryo transfer programme. Three ewes excreted chlamydiae at parturition 1 year after inoculation, with one animal exhibiting a CFT titre indicative of clinical disease. Twelve embryos collected from these three donors and transferred to seven disease-free recipients survived and were not infected, nor were their recipient dams. It therefore appears possible to infect ewe lambs during the final stages of pregnancy with the disease manifesting itself during the following breeding season. Embryos collected from infected animals do not appear to transmit the chlamydia causing enzootic abortion.

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.

Psittacosis pneumonia: a case report and review of the literature.

A case of acute, severe pneumonia with respiratory insufficiency due to Chlamydia psittaci is described. Rapid improvement with tetracycline therapy in all symptoms and arterial blood gases is demonstrated. The diagnosis of psittacosis was secured by both cultivation and serological verification. A comprehensive review of the literature is appended. The present report attests to the need for inclusion of C. psittaci as a possible aetiologic agent causing fulminating community-acquired pneumonia.

Chlamydia psittaci in turkeys: pathogenesis of infections in avian serovars A, B and D.

At 7 days of age, 4 groups, each of twenty specific pathogen free turkeys kept in isolation units were inoculated by aerosol with the Texas Turkey strain (avian Chlamydia psittaci serovar D), strain 92/1293 (avian Chlamydia psittaci serovar D), strain 84/55 (avian Chlamydia psittaci serovar A) or strain 89/1326 (avian Chlamydia psittaci serovar B). A fifth group of 4 specific pathogen free turkeys were sham inoculated controls. At daily intervals for 10 days and then twice weekly up to 34 days post infection, one bird in each group was killed and the target tissues and cells for replication and the sequence of events of serovar A, B and D infections was examined. In these turkeys, the primary site of replication was the respiratory tract. Chlamydial replication could be detected in the respiratory tract on day 1 post inoculation (p.i.) for group A, on day 3 p.i. for group B and on day 1 to 2 p.i. for groups D1 and D2. Subsequently, there was chlamydaemia and localisation in the digestive tract, in one or more parenchymatous organs, in the pericardium and in the conjunctivae. Specific immunoperoxidase staining revealed chamydiae in these organs in epithelial cells and in monomorphonuclear cells in all infected groups. The monomorphonuclear cells were identified as macrophages by double immunofluorescence staining. Chlamydiae were present in the same tissues for serovars A and D, but could not be demonstrated in proventriculus, duodenum, pancreas, ovaries and testes for serovar B. Furthermore, the intensity of replication was similar for all serovars. However, for serovar B in comparison with the other serovars, the bacteria appeared in most tissues 1 to 6 days later and the maximal replication in these tissues occurred 3 to 4 days later.

Diagnosis of ornithosis by cell culture and polymerase chain reaction in a patient with chronic pneumonia.

We report the case of a woman who had pneumonia due to Chlamydia psittaci. A Chlamydia species was determined to be the causative agent of the pneumonia because it was isolated from bronchoalveolar lavage fluid, because it could be detected in lung biopsy specimens by the direct immunofluorescence technique, and because Chlamydia-specific antibodies could be detected by ELISA and microimmunofluorescence. The infectious agent could not be identified at the species level with use of serological techniques, but the isolate was determined to be C. psittaci by PCR with use of species- and genus-specific sequences within the chlamydial lipopolysaccharide biosynthesis gene gseA. The case reported herein exemplifies the problems encountered in diagnosing ornithosis and shows that isolation of the etiologic agent followed by identification of the species by PCR is helpful in diagnosing this rare disease. In addition, the findings in our case show that laboratory personnel who are conducting tests for Chlamydia pneumoniae should be aware of the risk of accidentally isolating highly infectious C. psittaci organisms.

Respiratory chlamydia: the management of an outbreak.

This investigation was undertaken in response to the occurrence of nine cases of respiratory chlamydial infection in 8 months within the district of Dudley. All nine cases of respiratory chlamydia were due to Chlamydia psittaci, not Chlamydia pneumoniae. Five cases had avian exposure, but no other aetiological factors were identified. Faecal specimens were obtained from only two of the implicated birds and were negative. Two local aviaries were identified as sources of implicated birds. The two aviaries were themselves linked. Bird faecal specimens were taken from the two implicated aviaries and were both positive for Chlamydia psittaci. Appropriate public health control measures were introduced in these aviaries. A press statement was released to identify and advise, by telephone, those who purchased birds from the aviaries.

[Bronchoalveolar lavage fluid findings and lung function in psittacosis].

Three familial cases of psittacosis presented with fever and arthralgia. The first case was a 54-year-old man whose chest X ray film showed ground glass appearance in left S6. Ga scintigraphy revealed stronger accumulation in left lower lobe. Bronchoalveolar lavage fluid was examined in 2 of the cases and activated helper T cells were increased in both of the cases. Lung function was studied in all 3 cases in different phases. Their basic lung functions were different, but there was a similar change in courses, i.e. decrease of small airway flow and the increase of RV. These findings suggest that lung lesion of psittacosis may be related to allergic reaction and diffuse lung disease.

Psittacosis--a review of 135 cases.

We reviewed retrospectively 135 cases of serologically-confirmed psittacosis that were admitted to Fairfield Hospital between January 1, 1972 and March 31, 1986. The average age of the patients was 46 years. The majority (85%) of patients described a history of recent exposure to birds. The clinical features, investigations, treatment and subsequent response were analysed in 129 patients. Psittacosis was a well-defined illness that was characterized by an abrupt onset of fever, rigors, sweats, and prominent headache, and a mild dry cough which appeared late frequently. However, respiratory symptoms were absent in 18% of patients. Diarrhoea and sore throat were occasional complaints. Over 90% of cases had an abnormal chest x-ray film, or abnormal chest signs, or a combination of both. Most patients had a normal leukocyte count. Tetracycline drugs were used for treatment in 87% of the patients. Defervescence occurred in 92% of patients after 48 h of tetracycline treatment. There were no recrudescences of psittacosis and no fatalities. The clinical diagnosis of psittacosis can be made early usually, particularly in the presence of pneumonitis on a chest x-ray film and a positive history of bird contact. Treatment with doxycycline (100 mg twice a day for 14 days) is recommended.

Experimental chlamydial pneumonia in pigs.

Naturally occurring disease in pigs associated with chlamydial infections has not been reported in Britain, though evidence of chlamydial challenge has been demonstrated in two separate serological surveys. An isolate of Chlamydia psittaci (28/68) from an ovine pneumonia produced pneumonia in pigs following intratracheal inoculation. Transient pyrexia at 24 hr was followed by increased respiratory rates and inappetance which lasted for a further 48 hr in challenged pigs. Histologically acute exudative reactions were present in the lungs by 24 hr with proliferative changes predominating after 10 days. While variations in the concentrations of inocula were reflected by corresponding increases and/or decreases in gross lung damage, clinical signs and histological reactions were unaltered. Chlamydial organisms were recovered only from lung tissues.

Loss of inorganic ions from host cells infected with Chlamydia psittaci.

Mouse fibroblasts (L cells) infected with the 6BC strain of Chlamydia psittaci released potassium ion (K(+)) into the extracellular milieu in a way that depended on size of inoculum and time after infection. When the multiplicity of infection was 500 to 1,000 50% infectious units (ID(50)) per L cell, loss of intracellular K(+) was first apparent 4 to 10 h after infection and was nearly complete at 6 to 20 h. Magnesium ion and inorganic phosphate (P(i)) were also released. Similar multiplicities of ultraviolet-inactivated C. psittaci also caused release of K(+). Leakage of inorganic ions probably resulted from immediate damage to the host-cell plasma membrane during ingestion of large numbers of chlamydiae. With multiplicities of 1 to 50 ID(50) per L cell, ingestion of C. psittaci was not by itself enough to cause release of K(+) and P(i) from infected L cells. There was a delay of 36 to 72 h between infection and massive leakage of intracellular ions during which time the chlamydiae multiplied extensively. Fifty ID(50) of ultraviolet-inactivated C. psittaci per L cell did not bring about significant leakage of K(+), even after 72 h. The mechanism whereby these multiplicities of infection destroy the ability of host cells to retain intracellular molecules is not known. HeLa 229 cells also released K(+) and P(i) after infection, but these losses occurred more slowly than in comparably infected L cells, possibly because C. psittaci did not multiply as extensively in HeLa cells as it did in L cells. The significance of the inability of chlamydiae-infected cells to regulate the flow of molecules through their plasma membranes is discussed.