Chlamydophila Research Articles

Clinical characteristics analysis of patients with pneumonia infected by Chlamydia psittaci

To explore the clinical characteristics of pneumonia infected by Chlamydophila psittaci (C. psittaci). A retrospective analysis was performed on 3 cases of C. psittaci pneumonia admitted to People's Hospital of Tongling City from July 2019 to January 2020. The patients' contact history, clinical manifestations, laboratory examination, imaging characteristics and evolution, etiology, treatment process and outcome were analyzed, so as to provide experience for the diagnosis and prevention of C. psittaci pneumonia. The 3 patients had been infected through pet or zoonotic exposures. All symptoms included high fever (body temperature > 39 centigrade), cough, sputum, chest tightness and dyspnea. The disease progressed rapidly, with severe acute respiratory distress syndrome (ARDS) and shock as the main manifestations, but the damages to the heart, liver and kidney were mild. Laboratory tests showed that C-reactive protein (CRP, all > 200 mg/L) and neutrophil proportion (Neut%, > 0.90) were significantly increased, while white blood cell count (WBC) and procalcitonin (PCT) were not significantly increased. Chest computed tomography (CT) showed inflammatory infiltration with interstitial changes, either unilateral or bilateral. Chest X-ray showed large areas of inflammatory infiltrations, fan-shaped or wedge-shaped to the edge of pleura. After 7 days of treatment, the bedside computed X-ray (CR) showed absorption of infiltration. After 11-13 days, the CT reexamination indicated lung infection was basically absorbed. Metagenomic next-generation sequencing (mNGS) confirmed the presence of C. psittaci in patients' sputum. It was sensitive to quinolones and tetracyclines. The patients' body temperature dropped to normal after 2-3 days of antibiotics, and all patients were extubated and transferred to normal ward 10 days later. The total course of illness was 20-30 days. The patients with C. psittaci pneumonia are critically ill, and clinical manifestations of moderate to severe ARDS and shock are common. Early diagnosis depends on mNGS, and reasonable treatment is important for prognosis.

Diagnóstico molecular de neumonías bacterianas atípicas provenientes de la comunidad. Paraguay (2014-2017)

Objetivos: Determinar la frecuencia de neumonías bacterianas atípicas causadas por Legionella pneumophila, Mycoplasma pneumoniae y Chlamydophila pneumoniae, en niños y adultos de Paraguay durante el periodo 2014-2017 por técnicas moleculares.
 Materiales y métodos: se incluyeron en este estudio 148 muestras de líquido pleural de niños y adultos con diagnóstico de neumonía adquirida en la comunidad (NAC) durante el periodo 2014 a febrero de 2017. Las muestras fueron remitidas al Laboratorio Central de Salud Pública por los Centros Centinelas y Centros Colaboradores de la Red de Vigilancia de Meningitis y Neumonías.
 Resultados: La frecuencia general de neumonías atípicas resultó 1.4 % (2/148). Se detectó Legionella pneumophila en 0.7% (1/148) de los pacientes con sospecha de neumonía y un caso Mycoplasma pneumoniae en 0.7% (1/148).
 Conclusión: Los agentes atípicos presentan manifestaciones inespecíficas y variables de un caso a otro, haciéndolas difíciles de distinguir de otras etiologías. Los nuevos enfoques del diagnóstico microbiológico son prometedores, se espera que las pruebas de diagnóstico molecular para estos patógenos sean importantes herramientas en el laboratorio clínico.

Prevalence of antibodies to Chlamydophila abortus in ovines in the Londrina area of Paraná state, Brazil

O objetivo deste trabalho foi estimar a prevalencia de anticorpos anti- Chlamydophila abortus em ovinos de propriedades localizadas na regiao de Londrina, estado do Parana. A prova de fixacao de complemento foi realizada em 267 amostras de soro sanguineo de ovinos adultos, provenientes de oito propriedades e foram considerados positivos os soros com titulos ? 32. Em sete (87,50%) propriedades foram detectados 19 (7,11%) animais positivos, sendo 17 com titulos de 32, um com 64 e outro com 128. Nestas propriedades, os sinais clinicos descritos foram abortamentos, natimortalidade, nascimento de crias fracas e repeticao de cio. A elevada frequencia de propriedades com pelo menos um animal positivo e a ocorrencia de 19 (7,11%) animais positivos na prova de fixacao de complemento sugerem a presenca de Chlamydophila abortus nos animais das propriedades estudadas.

Immunogenicity analysis of a recombinant protein CPSIT_p7 and its expression in HeLa cells during persistent Chlamydophila psittaci infection

Objective To construct a prokaryotic expression plasmid for CPSIT_p7 gene from Chlamydophila psittaci （ Cps） 6BC strain and to evaluate immunogenicity of the recombinant protein His-CPSIT_p7 and detect its dynamic expression at mRNA and protein levels in HeLa cells during persistent Cps infection.Methods The fusion protein His-CPSIT_p7 was expressed in E.coli BL21 and purified by Ni-NTA affinity chromatography .BALB/c mice were immunized with the recombinant protein to prepare polyclonal antibody for evaluation of the immunogenicity of His-CPSIT_p7 by ELISA.Penicillin sodium was used to establish a model of Cps persistence infection .RT-PCR and Western blot assay were performed to de-tect the expression of CPSIT_p7 at mRNA and protein levels during Cps persistent infection .Results The fusion protein His-CPSIT_p7 was successfully expressed with the use of constructed recombinant expression plasmid pET30 a-CPSIT_p7 and purified .ELISA result showed that the specific antibody titer against CPSIT_p7 reached 1 ∶1 000 000 on the 40th days after immunization .The expression of CPSIT_p7 at mRNA and protein levels were increased in a time-dependent manner in Cps-infected HeLa cells .The peak of mRNA level was reached at the time point of 36 hours after infection , followed by a time-dependent decrease during Cps acute infection .However , the expression of CPSIT_p7 at mRNA and protein levels were not decreased until 60 hours after infection during Cps persistent infection .Conclusion His-CPSIT_p7 protein was suc-cessfully expressed in the prokaryotic expression system and purified , showing an advantage of good immuno-genicity.Highly expressed CPSIT_p7 at mRNA and protein levels were detected during Cps persistent infection. Key words: Chlamydophila psittaci; Cloning and expression; Immunogenicity; Per-sistent infection

Localization and temporal expression of CPSIT_0271 in Chlamydia psittaci-infected cells and its stimula-tory effects on the expression of proinflammatory cytokines by THP-1 cells

Objective To study the intracellular localization and temporal expression of CPSIT_0271 in Chlamydia psittaci-infected cells; and to investigate the effects of recombinant GST-CPSIT_0271 protein on the expression of proinflammatory cytokines including IL-6, IL-1βand TNF-αby THP-1 cells.Methods The gene encoding CPSIT_0271 of Chlamydia psittaci was expressed as fusion protein （ GST-CPSIT_0271 ） in E.coli.The polyclonal antibody was prepared by immunizing BALB /c mice with the purified recombinant pro-tein.Antibody titer was determined by ELISA .Indirect immunofluorescence assay （ IFA） was performed to lo-cate the endogenous CPSIT_0271 protein in C.psittaci-infected cells .The expression characteristics of CPSIT_0271 protein were detected by Western blot in C.psittaci-infected HeLa cells at different time points .The levels of IL-6, IL-1βand TNF-αwere analyzed by ELISA after stimulating THP-1 cells with different concentrations of CPSIT_0271 protein.Results CPSIT_0271 protein was found to express in the chlamydia inclusion of C.psittaci-infected HeLa cells .The expression of CPSIT_0271 protein was detected firstly at 36 h and increased at 48 h after C.psittaci infection.The titer of anti-CPSIT_0271 specific antibody in GST-CPSIT_0271 immu-nized mice reached to 1 ∶16 000.GST-CPSIT_0271 protein increased the levels of IL-6, IL-1βand TNF-αin THP-1 cells in a dose-dependent manner in the range of 2 to 5 μg/ml.The levels of TNF-αand IL-1βreached their peaks at 24 h, and IL-6 level peaked at 48 h upon the stimulation by 5 μg/ml of GST-CPSIT_0271 pro-tein.Conclusion CPSIT_0271 expressed in inclusion bodies of Chlamydia psittaci in the infected cells , sug-gesting it might be a late expression gene .GST-CPSIT_0271 protein shows good immunogenicity and enhances the expressions of IL-6, IL-1βand TNF-αin THP-1 cells. Key words: Chlamydophila psittaci; CPSIT_0271; Cellular localization; Temporal expression; Proin-flammatory cytokines

Secretion of TNF-α induced by heat shock protein 10 of Chlamydophila pneumoniae were mediated by TLR2 and TLR4 in THP-1

Objective To investigate the effect of heat shock protein 10 (HSP1O) of Chlamydophila pneumoniae in inducing TNF-α on THP-1 cells and the roles of TLR4 and TLR2 involved in it.Methods Purified native recombinant HSP10 from Cpn(CHSP10) were produced and inactivated the endotoxin contamination,then different concentration (0.5,1,5,10,20,30 μg/ml) of CHSP10 were used to stimulate THP-1 for different time (0,6,12,24,36,48,60 h).TNF-α were measured by using human TNF-α ELISA kit and compared among different groups.THP-1 were collected and analyzed for TLR2 and TLR4 mRNA levels and protein expression by RT-PCR and immunofluorescence.Peritoneal macrophages isolated from wide-type (C3 H/HeN) and TLR4-deficient mice (C3H/HeJ) were stimulated with endotoxin-free proteins respectively,and the TNF-α were measured.Furthermore,neutralizing anti-human TLR2/TLR4 McAb as a blocking Ab was preincubated with THP-1,after stimulation with CHSP10,ELISA was used to detect the concentration of TNF-α.Results TNF-α can be induced with CHSP10 in THP-1,while it significantly decreased with heated or deproteinized CHSP10.Both TLR2 and TLR4 mRNA and protein were detected in THP-1.Macrophages from C3H/HeN mice displayed higher TNF-α compared with it from C3H/HeJ mice after stimulation with CHSP10.The CHSP10-induced TNF-α would obviously decline when treated with antiTLR2/TLR4 McAb.Conclusion As a potential inflammation related protein,CHSP10 are involved in the pathogenesis of Cpn inducing inflammation cytokine TNF-α.TLR2 and TLR4 appear to be involved in CHSP10-mediated expression of TNF-α. Key words: Chlamydophila pneumoniae(Cpn) ; Heat shock protein 10; Toll like receptor; Tumor necrosis factor-α

Clinical and imaging features of neonatal chlamydial pneumonia

Objective To study the clinical and imaging features of chlamydial pneumonia in newborns.Methods Medical records,chest X-Ray and CT findings of 17 neonates with chlamydia pneumonia were reviewed.The age was ranged from 9.0 to 28.0 days with mean of ( 16.8 ± 5.8 ) days.There were 11males and 6 females.Sixteen were full term infants and one was born post term.All babies were examined with chest X-ray film,and 13 patients also underwent chest CT scan.Serologic test using immunofluorescence method for Chlamydia IgG and IgM antibodies were performed in all patients.Results All newborns presented with cough but without fever.Positive results of the serologic tests were demonstrated.Chest films showed bilateral hyperventilation in 10 patients,diffuse reticular nodules in 10 patients including nodules mimicking military tuberculosis in 7 patients,and accompanying consolidation in 9 patients.CT features included interstitial reticular nodules in 13 patients with size,density,and distribution varied.Subpleurul nodules ( 11patients) and fusion of nodules ( 10 patients ) predominated.Bilateral hyperinflation was found in 10 patients,which combined with infiltration in 12 patients,thickening of bronchovascular bundles in 10 patients,and ground glass sign in 5 patients.No pleural effusion and lymphadenopathy was detected in any patient.Conclusions Bilateral hyperinflation and diffuse interstitial reticular nodules were the most common imaging features of neonatal chlamydial pneumonia.The main clinical characteristic of neonatal chlamydial pneumonia is respiratory symptoms without fever,which is helpful to its diagnosis. Key words: Infant,newborn; Chlamydophila pneumoniae; Diagnostic imaging

Study on the diagnosis application using recombinant protein of immunodominant region gene of chlamydial protease-like activity factor from Chlamydophila psittaci

Objective To clone and express the immunodominant domain of the chlamydial proteaselike activity factor(CPAF) from Chlamydophila psittaci(Cps) and evaluated the diagnosing value of the recombinant protein in Cps infection.Methods The immunodominant region epitope of CPAF (CPAFm,A196-A450)from Cps was chosen according to bioinformatics analysis and references.The specific primer was designed and the gene was amplified by PCR and then ligated into a pGEX6p-2 vector.Recombinant protein was induced to express by IPTG and analyzed by SDS-PAGE and Western blot.Indirect EL1SA method of serological diagnosis was established with the reorganization protein as coating antigen.One hundred and eighty sera samples from ducks with respiratory tract infection symptoms were detected with the established indirect ELISA and a commercial ELISA-kit to assess the value of the recombinant protein in serodiagnosis.The results were further identified with Western blot.Results Prokaryotic expression vector pGEX6p-2/CPAFm was constructed and a 54x103 fusion protein was attained.The indirect ELISA method was established with reorganization protein for envelope antigen.Using the indirect ELISA to detect Cps lgG positive and negative reference sera,the sensitivity and specificity were both 100％ (20/20).And the recombinant protein has no cross reaction with either Chlamydophila pneumoniae or Chlamydophila trachomatis.The concordance rate between the indirect ELISA and Western blot to 180 ducks sera samples was 100％,while the concordance rate of the commercial ELISA kit was 77.5％-95.0％.Conclusion The prepared recombinant protein of the CPAF immunodominant region epitope gene from Gps can highly benefit on developing new indirect ELISA as methods to detect specific anti-Cps antibodies. Key words: Chlamydophila psittaci; Chlamydial protease-like activity factor; Immunocompetence; Serological diagnosis

Infecção por C. psittaci: uma revisão com ênfase em psitacídeos

A clamidiose ou ornitose é uma doença infecciosa, causada pela bactéria Chlamydophila psittaci, que acomete aves e mamíferos. Trata-se de uma das principais zoonoses de origem aviária. A transmissão ocorre principalmente por inalação de secreções contaminadas. Os sinais clínicos mais comuns incluem alterações no sistema gastrointestinal, respiratório e ocular, porém é possível encontrar aves infectadas sem sinais aparentes, dificultando a identificação da doença. O diagnóstico definitivo em aves vivas pode ser difícil, devido às características da infecção pela bactéria. Há duas principais abordagens para o diagnóstico, a primeira envolve a detecção direta da bactéria e a segunda implica a detecção de anticorpos anti-Chlamydophila sp. O tratamento é longo e envolve o uso de tetraciclinas, quinolonas ou macrolídeos, durante 21-45 dias, dependendo da espécie e do fármaco de escolha. Atualmente, o Brasil não dispõe de medidas padronizadas que visam a guiar o clínico na identificação, manejo e tratamento para a doença. Tais medidas tornam-se necessárias, bem como a pesquisa de novos métodos diagnósticos e auxiliares para a doença.

Is Dietary L-Lysine Effective for Treatment of Feline Ocular Disease Caused by Feline Herpesvirus-1?

Since the early 2000s, oral supplementation of L-lysine has been used to treat ocular disease in cats that was attributable to feline herpesvirus (FHV). Local (ocular) signs of disease that included conjunctivitis, blepharospasm, and ocular discharge were felt to improve when oral supplementation with a 250 or 500 mg bolus was administered once to twice daily. FHV-1 is considered the most common pathogen among other infectious agents, i.e., feline calicivirus and Chlamydophila felis, to cause ocular disease as well as infectious upper respiratory disease. It is felt that FHV-1 may establish a lifelong neural latency in 80% of infected cats. Early studies proposed that oral bolus administration of L-lysine reduced replication of FHV-1 by approximately 80% via antagonism of arginine, an essential amino acid for FHV-1 replication. Arginine is an essential dietary need for normal cat immune function. Studies evaluating serum arginine levels following oral L-lysine dose trials have found no adverse diminished levels of arginine. L-lysine is commercially available over the counter for treatment of the common cold sore in people that is caused by herpes simplex virus type 1. This formulation may be used for treatment of feline ocular disease by administering pills orally or by adding the contents of capsules to the diet. The generally recommended dose is a bolus of 500 mg, per os, twice per day in adult or average sized cats. Younger or smaller cats are typically fed 250 mg, twice per day. It has been shown that diets fortified with L-lysine that are consumed gradually throughout the day may not provide the immunologic effect necessary to reduce ocular disease and, specifically, infectious upper respiratory disease. Since the emergence of L-lysine as an effective and safe, long-term therapeutic for FHV-1 treatment, a number of commercially available products have become available on the veterinary market. By using palatable gels and treat products, administering a consistent and effective dosage regimen has become less problematic.

Chlamydial protease-like activity factor from Chlamydophila pneumoniae induced THP-1 cells produced proinflammtory cytokines and apoptosis

Objective To express and purify Chlamydial protease-like activity factor(CPAF)from Chlamydophila pneumoniae,for investigating the effect of its recombinant protein GST-CPAF in inducing human monocytic cells to secrete proinflammatory cytokines and cell apoptosis.Methods The recom-bination expression plasmid pGEX6p-2/CPAF from Chlamydophila pneumoniae was transformed into E.coli.The recombination GST-CPAF was expressed after induction by IPTG,and purified by a agarose gel FF.Human monocytic cells were stimulated by the GST-CPAF to test the production of tumor necrosis factor a(TNF-α)and interleukin-6(IL- 6)by ELISA.Inhibition of cells proliferation with GST-CPAF was assessed by MTT.The THP-1 cell apoptosis stimulated by GST-CPAF was detected by Hoechst33258 fluorescence staining,DNA fragmentation analysis and cell apeptosis was detested bv Annexin V-FITC-propidiuum iodide (PI)staining.Results The recombination protein GST-CPAF was successfully expressed with high level in E.coli,and stimulated human monocytic cells to produce proinflammatory cytokines including TNF-α and IL-6 in a dose-and time-dependent manner.Otherwise,the GST-CPAF inhibited the growth of human monocytic cell in a dose-dependent manner.Apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent staining and microscopy,DNA ladders in apoptosis cells were detected after 24 h with the GST-CPAF.Conclusion The GST-CPAF from Chlamydophila pneumoniae can induce the secretion of proinflammatory cytokines TNF-α and IL-6 by human monocytic cells,and inhibited the proliferation of THP-1 cell and apoptosis in vitro. Key words: Chlamydophila pneumoniae; Chlamydial protease-like activity factor; THP-1; Proinflammatory cytokines; Cell apoptosis

Comparison of three different novel Chlamydophila pneumonia recombinant antigens for serodiagnosis

Objective To evaluate three different Chlamydophila pneumoniae recombinant antigens for use in Chlamydophila pneumoniae serodiagnosis. Methods The recombinant plasmids pGEX6p-2/ Cpn0146,Cpn0147 and Cpn0308 were constructed and expressed as GST fusion proteins. The immunogenicity and the immunocompetence of these recombinant protein were analyzed by Western-blot and indirect ELISA. A total of 183 sera samples of patients with respiratory tract infection and 32 sera samples of patients with Chlamydia trachomatis infection were detected with indirect ELISA coated microwell plates with the purified recombinant proteins comparing with SeroCP-TM IgG ELISA kits. The positive recognition rate, sensitivity and specificity of each method were analyzed. Results GST-Cpn0146, Cpn0147 and Cpn0308 were obtained after expression and purification. The titers of the specific IgG antibodies against Cpn0146, Cpn0147 and Cpn0308 were higher than 1:6 400, 1:128 00 and 1:128 00, respectively. When the indirect ELISA was developed to detect the IgG antibody against Chlamydophila pneumoniae in 183 samples, the concordance rate between the indirect ELISA test and SeroCP-TM IgG ELISA kits were 92. 3% (Cpn0146) , 94.5% (Cpn0147) and 96.7% (Cpn0308), respectively. The recombinant Cpn0146, Cpn0147 and Cpn0308 were recognized by 71 (38.8% positive recognition rate), 75 (40.9%), and 82 (44.8%) samples, respectively. The recombinant antigen-based detection assays displayed > 97% of detection specificity and>87%of sensitivity.Condusion GST-Cpn0308 shows a better sensitivity and specificity,which suggests it could be used for developing serodiagnosis kits of Chlamydophila pneumoniae infection. Key words: Chlamydophila pneumoniae; Antigens,bacterial; Recombinant proteins; Serologic tests

Unanticipated outbreak of Q fever during a study using sheep, and its significance for further projects

Effective measures for protection of laboratory staff against infections due to experiments with pathogens are a challenge for safeguarding against hazards. Investigations on limited epidemic infections and their causes are of high relevance. During an experiment with pregnant sheep 5 of 7 persons who took part in the operation procedures developed Q fever 10 to 24 days after exposure. One sheep aborted spontaneously 3 days after the third experiment. Examinations of the aborted material revealed Coxiella burnetii, Chlamydophila psittaci, and Staphylococcus chromogenes. Coxiella burnetii and Chlamydophila psittaci were also detected in some of the other placentae. This outbreak of Q fever during an experiment with infected sheep affected more than 70% of the people involved with this experiment and shows the high risk of working with the zoonotic agent Coxiella burnetii. Hence, technical, organizational, and personnel measures have to be taken, and laboratory animals are to be monitored to prevent further infections.

In vitro activities of demestic macrolides against Chlamydia trachomatis and Chlamydophila pneumoniae

Objective To evaluate the activities of four demestic macrolides against C. trachomatis and C. pneumoniae by antimicrobiai susceptibility testing. Methods Cell culture and immunoflourescence staining of chlamydial inclusions were used to determine MICs of four demestic macrolides against C. trachomatis and C. pneumoniae. Results MIC (0.5 μg/ml) was found for acylspriramycin,erythromycin and azithromycin against C. trachomatis serovar B while it was 4 μg/ml for acetylspiramycin. Agaisnt C. trachomatis serovar D, MIC was 0.25 μg/mi in both acylspriramycin and azithromycin, and MICs were 0.5 μg/ml and 2 μml in erythromycin and acetylspiramycin, separately. Agaisnt C. pneumoniae TWAR, erythromycin was the most active with MIC≤0. 016 μg/ml, acylspriramycin and azithromycin were the second with same M1C of 0.032 μg/ml. However, acetylspiramycin was less active with 0.5 μg/ml of MIC. Conclusion Except acetylspiramycin, acylspriramycin erythromycin and azithromycin had reliable activities against both C. trachomatis (serovar B and D) and C. pneumoniae. Key words: Chlamydia trachomatis; Chlamydophila pneumoniae; Macrolides; Microbial sensitivity tests