| Chlamydia spp. are the obligate intracellular gram-negative bacterium with unique developmental cycle of elementary body and reticulate body, which are widely exist in nature and leading to outbreaks of chlamydiosis in multiple animals and humans. The family Chlamydiaceae with its single genus Chlamydia (C.) currently contains 11 recognized species, namely C. trachomatis, C. suis, C. muridarum, C. pneumoniae, C. abortus, C. caviae, C. felis, C. pecorum, C. psittaci, and two recently introduced species, C. avium and C. gallinacea. Chlamydial infections usually cause tremendous economic losses to the bird owners and breeding industry, which also result in enormous influences on public health security for import and export trade of animal products and potential zoonotic transmission to human.C. psittaci has been considered to be the sole causative agent of avian chlamydiosis for decades. The epidemic data of Chlamydia spp. in poultry of China is limited, and the knowledge of pathogenicity and genomics of C. avium and C. gallinacea is poorly understood worldwide. This project systematically studied the prevalence status of Chlamydia spp. in poultry of China (chickens, ducks, geese and pigeons), and researched the pathogenicity and genomics of C. gallinacea JX-1 isolated in this paper.1. Establishment of FRET-qPCR for detection of 11 Chlamydia spp.The general detection methods for Chlamydia spp. include isolation and culture, direct staining microscopy, indirect hemagglutination test (IHA), complement fixation test (CFT), enzyme linked immunosorbent assay (ELISA), PCR, etc. The traditional method of isolation and culture has widely been regarded as the gold standard for Chlamydia spp. dection, but which is time consuming and the sensitivity is limited; ELISA with high specificity and sensitivity would yet induces cross reaction with other microorganisms; the facility request of IHA and CFT is low, but possessing the complex operation and low sensitivity. Serology methods have frequently been applied for the detection of batch specimens, however, they will probably cause false positive results as they are unable to differentiate immunization and natural infection.Following the continuous development of Chlamydia spp. detecting techniques, there are more and more detection methods, while novel molecular biology method is the new development trend for pathogen detection. There are many PCR methods applied for chlamydial detection, including general PCR, multiplex PCR, nested PCR, real-time fluorescence quantitative PCR. Fluorescent quantitative PCR method is a real time quantitative determination technology for specific nucleotides with high specificity and sensitivity, which has been widely used in various Chlamydia spp. detection, such as C. pneumoniae, C. trachomatis, C. psittaci, C. abortus, C. pecorum, C. felis, C. avium and C. gallinacea. PCR methods established currently only target for one or several Chlamydia species, yet could not able to detect multiple Chlamydia species simultaneously, so which is very inconvenient for chlamydial detection from multiple infections of clinical specimens. Therefore, this study established a FRET-qPCR method for detecting all 11 Chlamydia species simultaneously based on 23 S rRNA gene of Chlamydia spp., which was able to differentiate them into eight groups depending on the melting temperature generated by Chlamydia spp., and need further confirmatory sequencing for Chlamydia species with close melting temperature. The FRET-qPCR method established here with high specificity, sensitivity and well repeatability was the first FRET-qPCR method wordwide established for simultaneously detecting and almost differentiating all 11 Chlamydia spp.2. Research on the diversity of Chlamydia spp. in poultry of ChinaA total of 3,962 oral and cloacal swabs were collected from 2,300 apparently healthy poultry (1,791 chickens,215 pigeons,179 ducks and 115 geese) in live-bird parkets in 24 provinces of China in this project; oral and cloacal swabs of 1,662 birds were both obtained, but in some cases only oral swabs (n=476) or cloacal swabs (n=162) were collected. FRET-qPCR established in this study, combined with high-resolution melting curves and further DNA sequencing analysis were used to identify the Chlamydia species. Results showed that total positivity of Chlamydia spp. in poultry was 26.2%(602/2,300), and the highest positivity was in pigeon, reached 49.3%(106/215), followed by chickens 24.7%(442/1,791), ducks 24.0% (43/179) and geese 9.6%(11/115). The prevalence of Chlamydia spp. showed considerable geographic differences:the prevalences were above 30% in nine provinces, and the highest was 80.0%(56/70) in Sichuan; Chlamydia spp. were not detected in five provinces, including Shanxi, Tibet, Jilin, Liaoning and Shanghai; while the prevalence ranged from 1%-26% in ten other provinces. Overall, the positivity of oral swabs (22.0%) was significantly higher than that of cloacal swabs (16.1%). In birds from which both oral and cloacal swabs were collected (1,662), the positivity of Chlamydia spp. was 27.3% and 162 poultry were positive in both swabs. Of the 162 Chlamydia positive birds in both swabs,142 harbored the same species and the other 20 poultry with inconsistent Chlamydia spp. representing double infection.This study identified five Chlamydia species from poultry of China totally:C. gallinacea, C. psittaci, C. pecorum, C. suis and C. muridarum. The most popular chlamydial species were C. gallinacea and C. psittaci, which were detected from four kinds of poultry; the former accounted for 61.7%(384/622) in Chlamydia positive poultry and mainly found in chickens, while the latter represented 24.0%(149/622) and mostly originated from pigeons. In addition, C. pecorum was detected in three oral swabs of chickens from Inner Mongolia, and C. suis and C. muridarum were accounted for 10.5%(65/622) and 3.4%(21/622) among the Chlamydia positive poultry separately. The detection of C. suis and C. muridarum from poultry in this research is the first report worldwide, and indicated that C. gallinacea was the prevalent Chlamydia species in poultry, yet C. psittaci was only popular in pigeons.3. Dynamic monitoring of three-yellow chickens naturally infected with Chlamydia spp.Studying of Chlamydia spp. diversity in poultry of China showed multiple Chlamydia species existed in healthy poultry from live-bird markets, and found four Chlamydia species in three-yellow chickens from a mountain village in Xingan county of Jiangxi province, including C. psittaci, C. gallinacea, C. suis and C. muridarum. In this study, three-yellow chickens originated from the mountain village were conducted for chlamydial dynamic monitoring of seven months and sampling for nine times. The first three samplings were in the mountain village and the last six samplings proceeded when chickens were being transferred to a containment animal facility in Jiangsu Institute of Poultry Science. The positivity of Chlamydia spp. in chickens were 74.4%(35/57) and 40.9%(36/88) respectively at the first two detections; thirty-one randomly selected Chlamydia-positive chickens transferred to Jiangsu Institute of Poultry Science after the third sampling, which naturally infected with C. suis (28/31; 90.3%), C. psittaci (2/31; 6.5%) and C. gallinacea (1/31; 3.2%). Among four Chlamydia species detected in the first three samplings, C. suis was the mainly prevalent species and occupied 88.6%(31/35), 83.3%(30/36) and 90.3%(28/31) respectively in Chlamydia positive chickens.The positivity of Chlamydia spp. in chickens decreased remarkably in the fourth to fifth samplings after being transferred to Jiangsu Institute of Poultry Science. C. suis and C. psittaci had disappeared completely in the sixth to ninth detections, while C. gallinacea became the major popular species gradually as the only detectable species, and the carrier status was persisting until the end of monitoring. Furthermore, five chickens were randomly selected and euthanized for chlamydial detection, which showed that C. gallinacea was detected from whole blood, oral and cloacal swabs, and other seven organs, including trachea, heart, liver, spleen, lung, kidney and pancreas. Dynamic monitoring results demonstrated that C. suis and C. psittaci had gradually disappeared along with the changed environment, while C. gallinacea increased and became the mainly prevalent species. Hereby speculated that the infection of C. suis and C. psittaci in three-yellow chickens were probably only transient, but C. gallinacea maybe the commensal in natural host of chicken.4. Isolation and identification of Chlamydia gallinacea and Polymorphism analysis of ompAIn this research, four C. gallinacea isolates (JX-1, JX-2, JX-3 and JX-4) were successfully isolated from an oral swab and three cloacal swabs of C. gallinacea positive three-yellow chickens by Hep-2 cells. The ompA gene sequences (GenBank accession number:KT692977) of these isolated strains were identical to each other and showed 85.7% similarity to corresponding ompA gene sequences available in GenBank (accession number:AWUS01000004).C. gallinacea positive swabs identified in this study were conducted for PCR amplification and sequencing based on the variable domains 1-2 and 3-4 of ompA gene, and constructed the phylogenetic tree through multiple comparative analyses between the sequencing results and the ompA sequences in GenBank. Phylogenetic analysis showed that the ompA gene of C. gallinacea was highly polymorphic, and at least 13 new serotypes deeply separated were identified in addition to the published ompA sequences in GenBank. Nowadays, there is only one C. gallinacea strain successfully isolated from France in the world; yet four C. gallinacea strains were isolated from three-yellow chickens of Jiangxi province in this research, which is of great significance for further research of the pathogenicity, host range, spread mechanism and the potential zoonotic probability of C. gallinacea.5. Influence of Chlamydia gallinacea inoculation on chick embryos and growth of broilersSPF chick embryos and AA broilers were inoculated with C. gallinacea strain JX-1 isolated in this study to observe the influences of C. gallinacea on pathogenicity for chick embryos and gain weight for broilers. Chick embryo inoculation test was divided into 3 experimental groups and one control group with 5 embryos in each group. Three different dosages of C. gallinacea in high, medium and low inoculation groups were 105,104,103 genomes/μl respectively. Chick embryos of experimental groups were inoculated with 200 μl C. gallinacea Strain JX-1 through yolk sac at 7 days old, while chick embryos in control group were infected with 200 μl SPG buffer. Results showed that chick embryos were all died at 10 to 18 days old erratically, and the lesions of embryos especially in high dosage group were severity, appeared spheric and curled up, systemic hemorrhage, such as hemorrhage and necrosis of liver, hemorrhage of pericardium and intestinal tract; distinct concentration of C. gallinacea were detected from yolk sac membrane, yolk and allantoic fluid of chick embryos by FRET-qPCR, and the highest was in yolk sac membrane (2×107 genomes/mg), followed by yolk (4×103genomes/μl) and allantoic fluid (2×103 genomes/μl). While chick embryos in control group were growing until hatch out of their shells.AA broilers of 7 days old were inoculated with 20μl C. gallinacea strain JX-1 (105 genomes/μl) through intranasal. Results showed that chickens of the experimental and control groups were not showing any clinical symptoms. Swabs collected from chickens were Chlamydia spp. negative by FRET-qPCR detection at 7 days old before inoculation, and there was no significant difference in body weight of broilers between the experimental and control groups; After inoculation, there was still no significant difference in two groups at the first two weeks, but at the third to the fifth weeks, body weight of the experimental group was significantly lower than that of control group (883 ± 54 [SEM] vs.962 ± 61 g,1165 ± 80 vs. 1315±93 g,1539±120 g vs.1645±133 g). As well, there was no significant difference in weekly gain weight of chickens between two groups at the first two weeks, and weekly gain weight of the experimental group was lower than that of control group by 2.9% and 5.2%; however, weekly gain weight of the experimental group was significantly lower than that of control group (8.2%,11.4%,6.5%; P< 0.0009) at the third to the fifth week. Thus it could be seen that the pathogenicity of C. gallinacea JX-1 for chick embryo was high, and the gain weight of broiler was significantly influenced by C. gallinacea JX-1, although there was no symptom.6. Whole-genome sequencing and comparative genomics of C. gallinacea JX-1C. gallinacea JX-1 isolated in this study was conducted whole-genome sequencing by the third generation technology. Therefore, we obtained the second whole-genome sequence of C. gallinacea worldwide. The genome of C. gallinacea JX-1 is composed of a 1.05 Mbp with an overall GC content of 37.93%; 957 genes were identified accounting for 91.21% of the genome; ncRNA including 39 tRNAs, two copies of 5S rRNA, and single copy of 16S rRNA and 23S rRNA; possess of 24 tandem repetitive domains,37 scattered repetitive sequences and one gene island of 10,815 bp; but lack of prophage, short palindrome repetitive sequences and secondary metabolism gene clusters.Encoded genes and functional annotation of their corresponding products were obtained by comparative analysis of C. gallinacea JX-1 encoding sequences in authoritative database. Annotation results showed that there are 625 related genes in GO database,576 in KEGG database,644 of 26 categories in COG database and 469 in Swiss-Prot database. There are 26 predicted secretory proteins,6 proteins of T2SS and 39 proteins of TTSS; among 61 predicted effective proteins of TTSS, there are 2 effective proteins encoded by genes GM000184 and GM000192, which locate in the single gene island. Annotation of 17 phenotypic mutation genes in PHI database demonstrated that 12 genes are related to reduced virulence and the only gene GM000417 correlate with pathogenicity loss. There are 29 proteins of T3SS and a protein of T2SS from 38 virulence factors annotated in VFDB database, while the other 8 virulence factors are essentially consistent with C. gallinacea reference sequences. Comparative genomics of C. gallinacea JX-1 with 11 Chlamydia spp. showed that, the similarity of C. gallinacea JX-1 with 99.99% coverage and C. gallinacea reference strain with 99.29% coverage are 99.33%-99.58%, and the identity of C. gallinacea JX-1 with 96.85% coverage and C. avium reference strain with 97.97% coverage are 53.06%-88.68%, however, coverage of the other 9 Chlamydia species are less than 40%, and the similarity of 5.56%-37.24% showing greater discrepancy. Thus, the collinearity of C. gallinacea JX-1 and C. gallinacea reference genomes is preferable while more discrepancy with other Chlamydia species. However, SV analysis of these two C.gallinacea strains showed that strain JX-1 have one insertion of 126 bp and one ComplexInDel area in reference strain; moreover, the significant discovery is that, the similarity of some virulence factors between bacteria fragile and C. gallinacea JX-1 are highly identity, even consistent, which indicated that mutual virulence factors emerged as bacterial evolution may play an important role in the pathogenicity.In conclusion, Chlamydia spp. are zoonotic pathogens infected with multiple animals and humans, widespread world widely with broad host range, which have been overlooked frequently as clinical symptoms mostly are not obvious. C. psittaci has usually been considered as the only species infecting poultry for long time, while five Chlamydia species were detected from poultry in this study, which completely changed the previous opinion. C. gallinacea was the predominant Chlamydia species in poultry, and the detection of C. suis and C. muridarum from poultry was the first report in the world. Moreover, four C. gallinacea strains (JX-1, JX-2, JX-3 and JX-4) were successfully isolated from poultry; animal experiments showed that the pathogenicity of C. gallinacea for chick embryo was powerful, and the impact of C. gallinacea for the gain weight of broilers decreased significantly, reaching 11.4%. In addition, the third generation technology was adopted for whole-genome sequencing of C. gallinacea XX-1, thus second of C. gallinacea whole-genome sequence of C. gallinacea worldwide were obtained. The collinearity of C. gallinacea JX-1 and C. gallinacea reference genomes was preferable, but the SV analysis of these two C. gallinacea strains showed that there were one insertion of 126 bp and one ComplexInDel area in the reference strains; moreover, the significant discovery was that, the similarity of some virulence factors between the bacteria fragile and C. gallinacea JX-1 was highly identity, almost the same. Therefore, the investigation upon the diversity of Chlamydia spp. in Poultry and genomics of Chlamydia gallinacea were beneficial for the study of Chlamydia spp. prevalence, genetic variation and pathogenic mechanism, zoonotic probably, and the interaction of pathogen and host, also establish the theory and reference basis for further mastery of the functional genomics discrepancy and the transcriptomics and proteomics of Chlamydia spp. research. |
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