Whole genome sequencing characteristics of Chlamydia psittaci caprine AMK-16 strain, a promising killed whole cell veterinary vaccine candidate against chlamydia infection.

Chlamydia psittaci is a primary zoonotic pathogen with a broad host range causing severe respiratory and reproductive system infection in animals and humans. To reduce the global burden of C. psittaci-associated diseases on animal welfare and health and to control the pathogen spread in husbandry, effective vaccines based on promising vaccine candidate(s) are required. Recently, the caprine C. psittaci AMK-16 strain (AMK-16) demonstrated a high level of protection (up to 80-100%) in outbred mice and pregnant rabbits immunized with these formaldehyde-inactivated bacteria against experimental chlamydial wild-type infection. This study investigated the molecular characteristics of AMK-16 by whole-genome sequencing followed by molecular typing, phylogenetic analysis and detection of main immunodominant protein(s) eliciting the immune response in mouse model. Similarly to other C. psittaci, AMK-16 harbored an extrachromosomal plasmid. The whole-genome phylogenetic analysis proved that AMK-16 strain belonging to ST28 clustered with only C. psittaci but not with Chlamydia abortus strains. However, AMK-16 possessed the insert which resulted from the recombination event as the additional single chromosome region of a 23,100 bp size with higher homology to C. abortus (98.38-99.94%) rather than to C. psittaci (92.06-92.55%). At least six of 16 CDSs were absent in AMK-16 plasticity zone and 41 CDSs in other loci compared with the reference C. psittaci 6BC strain. Two SNPs identified in the AMK-16 ompA sequence resulted in MOMP polymorphism followed by the formation of a novel genotype/subtype including three other C. psittaci strains else. AMK-16 MOMP provided marked specific cellular and humoral immune response in 100% of mice immunized with the inactivated AMK-16 bacteria. Both DnaK and GrpE encoded by the recombination region genes were less immunoreactive, inducing only a negligible T-cell murine immune response, while homologous antibodies could be detected in 50% and 30% of immunized mice, respectively. Thus, AMK-16 could be a promising vaccine candidate for the development of a killed whole cell vaccine against chlamydiosis in livestock.

Protective Immunity against Chlamydia psittaci Lung Infection Induced by a DNA Plasmid Vaccine Carrying CPSIT_p7 Gene Inhibits Dissemination in BALB/c Mice.

Chlamydia psittaci (C. psittaci), a zoonotic pathogen, poses a potential threat to public health security and the development of animal husbandry. Vaccine-based preventative measures for infectious diseases have a promising landscape. DNA vaccines, with many advantages, have become one of the dominant candidate strategies in preventing and controlling the chlamydial infection. Our previous study showed that CPSIT_p7 protein is an effective candidate for a vaccine against C. psittaci. Thus, this study evaluated the protective immunity of pcDNA3.1(+)/CPSIT_p7 against C. psittaci infection in BALB/c mice. We found that pcDNA3.1(+)/CPSIT_p7 can induce strong humoral and cellular immune responses. The IFN-γ and IL-6 levels in the infected lungs of mice immunized with pcDNA3.1(+)/CPSIT_p7 reduced substantially. In addition, the pcDNA3.1(+)/CPSIT_p7 vaccine diminished pulmonary pathological lesions and reduced the C. psittaci load in the lungs of infected mice. It is worth noting that pcDNA3.1(+)/CPSIT_p7 suppressed C. psittaci dissemination in BALB/c mice. In a word, these results demonstrate that the pcDNA3.1(+)/CPSIT_p7 DNA vaccine has good immunogenicity and immunity protection effectiveness against C. psittaci infection in BALB/c mice, especially pulmonary infection, and provides essential practical experience and insights for the development of a DNA vaccine against chlamydial infection.

Chlamydia psittaci plasmid-encoded CPSIT_P7 induces macrophage polarization to enhance the antibacterial response through TLR4-mediated MAPK and NF-κB pathways.

Although the protective effects of Chlamydia psittaci plasmid-encoded protein CPSIT_P7 as vaccine antigens to against chlamydial infection have been confirmed in our previous study, the function and mechanism of CPSIT_P7 inducing innate immunity in the antibacterial response remain unknown. Here, we found that plasmid protein CPSIT_P7 could induce M1 macrophage polarization upregulating the genes of the surface molecule CD86, proinflammatory cytokines (TNF-α, IL-6, and IL-1ß), and antibacterial effector NO synthase 2 (iNOS). During M1 macrophage polarization, macrophages acquire phagocytic and microbicidal competence, which promotes the host antibacterial response. As we observed that CPSIT_P7-induced M1 macrophages could partially reduce the infected mice pulmonary Chlamydia psittaci load. Furthermore, CPSIT_P7 induced M1 macrophage polarization through the TLR4-mediated MAPK and NF-κB pathways. Collectively, our results highlight the effect of CPSIT_P7 on macrophage polarization and provide new insights into new prevention and treatment strategies for chlamydial infection.

A recombinant multi-epitope peptide vaccine based on MOMP and CPSIT_p6 protein protects against Chlamydia psittaci lung infection.

Chlamydia psittaci is an obligate intracellular pathogen with a broad host range that can lead to severe infectious disease by transferring from birds to humans. Vaccination has been considered the best way to prevent chlamydial infection; nevertheless, there is currently still no commercially available vaccine that can inhibit the spread of C. psittaci. In previous study, major outer membrane protein (MOMP) of C. psittaci was confirmed to be an appropriate candidate antigen for limiting C. psittaci respiratory infections in a murine model, and plasmid-encoded CPSIT_p6 also has functions similar to those of MOMP in our study. Therefore, according to bioinformatics analysis, we developed a recombinant peptide containing multiple antigenic epitopes from MOMP (24-32, 262-272) and CPSIT_p6 protein (109-119, 173-181) and evaluated the efficacy of peptide immunization. BALB/c mice were inoculated intraperitoneally with the recombinant multi-epitope antigens three times at 2-week intervals and subsequently intranasally infected with C. psittaci. We found that the recombinant multi-epitope antigens induced strong humoral and Th1 cellular immune responses by producing meaningfully high levels of antigen-specific antibodies, interferon-gamma (IFN-γ), or interleukin-2 (IL-2). Vaccination significantly reduced the bacterial burden and the degree of inflammation in the infected lungs and led to lower levels of IFN-γ and IL-6. Furthermore, adoptive transfer of CD4+ splenocytes harvested from the vaccinated mice produced a significantly lower chlamydial load, indicating the importance of the cellular immune response. Therefore, the recombinant multi-epitope antigens may provide the basis for a new peptide-based vaccine against C. psittaci infection.

Intersectoral action for health: preventing psittacosis spread after one reported case

Zoonotic diseases are a significant health threat for humans and animals. To better understand the epidemiology, etiology, and pathology of infectious agents affecting humans and animals combined approaches are needed. Here we describe an epidemiological investigation conducted by physicians and veterinarians after a reported case of psittacosis. Upon admission suffering from respiratory distress syndrome in a hospital and with a history of bird contact, a female patient was serologically diagnosed with psittacosis. After the case notification, veterinarians were able to investigate the source of infection by detecting Chlamydia psittaci in her pet cockatiel. The bird was hospitalized and successfully treated. In addition, the establishment where the pet bird was purchased was traced and through molecular techniques other birds intended to be sold as pets tested positive for C. psittaci. As a result, sanitary measures were applied and the establishment then was closed down. The birds intended for the pet commerce were treated and retested with negative molecular results for C. psittaci, thus avoiding disease propagation. Reliable data about zoonotic diseases can only be generated through the application of multidisciplinary approaches which take into account the epidemiological factors and interactions of humans, animals and their environments as an integrated system

Compendium of Measures to Control Chlamydia psittaci Infection Among Humans (Psittacosis) and Pet Birds (Avian Chlamydiosis), 2017.

Psittacosis, also known as parrot fever and ornithosis, is a bacterial infection that can cause severe pneumonia and other serious health problems in humans. It is caused by Chlamydia psittaci. Reclassification of the order Chlamydiales in 1999 into 2 genera (Chlamydia and Chlamydophila) was not wholly accepted or adopted. This resulted in a reversion to the single, original genus Chlamydia, which now encompasses all 9 species including Chlamydia psittaci. During 2003-2014, 112 human cases of psittacosis were reported to the Centers for Disease Control and Prevention through the Nationally Notifiable Diseases Surveillance System. While many types of birds can be infected by C psittaci, in general, the literature suggests that human cases can most often occur after exposure to infected parrot-type birds kept as pets, especially cockatiels, parakeets, and conures. In birds, C psittaci infection is referred to as avian chlamydiosis. Infected birds shed the bacteria through feces and nasal discharges, and humans become infected from exposure to these materials. This compendium provides information about psittacosis and avian chlamydiosis to public health officials, physicians, veterinarians, the pet bird industry, and others concerned with controlling these diseases and protecting public health. The recommendations in this compendium provide standardized procedures to control C psittaci infections. This document will be reviewed and revised as necessary, and the most current version replaces all previous versions. This document was last revised in 2010. Major changes in this version include a recommendation for a shorter treatment time for birds with avian chlamydiosis, additional information about diagnostic testing, including genotyping, clearer language associated with personal protective equipment recommended for those caring for confirmed or exposed birds, and incorporating a grading scale with recommendations generally based on the United States Preventive Services Task Force's methods.

Intersectoral action for health: preventing psittacosis spread after one reported case.

Zoonotic diseases are a significant health threat for humans and animals. To better understand the epidemiology, etiology, and pathology of infectious agents affecting humans and animals combined approaches are needed. Here we describe an epidemiological investigation conducted by physicians and veterinarians after a reported case of psittacosis. Upon admission suffering from respiratory distress syndrome in a hospital and with a history of bird contact, a female patient was serologically diagnosed with psittacosis. After the case notification, veterinarians were able to investigate the source of infection by detecting Chlamydia psittaci in her pet cockatiel. The bird was hospitalized and successfully treated. In addition, the establishment where the pet bird was purchased was traced and through molecular techniques other birds intended to be sold as pets tested positive for C. psittaci. As a result, sanitary measures were applied and the establishment then was closed down. The birds intended for the pet commerce were treated and retested with negative molecular results for C. psittaci, thus avoiding disease propagation. Reliable data about zoonotic diseases can only be generated through the application of multidisciplinary approaches which take into account the epidemiological factors and interactions of humans, animals and their environments as an integrated system.

Recombinant protein CPSIT_0846 induces protective immunity against Chlamydia psittaci infection in BALB/c mice.

Chlamydia psittaci is an obligate intracellular bacteria that causes respiratory disease in poultry and humans. Currently, there are no licensed vaccines against chlamydial infection in humans. The transmembrane head protein CPSIT_0846 of C. psittaci is a putative member of the larger Inc protein family. In this study, we investigated immunogenicity and protective efficacy of the recombinant CPSIT_0846 protein in BALB/c mice. Mice immunized with CPSIT_0846 developed strong T-lymphocyte responses that were recalled by the immunogen CPSIT_0846 in an in vitro restimulation assay. These T cells displayed a strong Th1-biased cytokine profile with high levels of IFN-γ. At the same time, a strong humoral immune response was also detected in the immunized mice with high titers of Chlamydia psittaci-specific serum IgG antibodies. More importantly, the robust immune responses correlated well with significantly reduced chlamydial burden and inflammatory pathology in the mouse lungs upon an airway challenge infection. The above results together suggest that the CPSIT_0846 protein may be a potential vaccine candidate antigen for inducing protection against C. psittaci infection and disease in the airway.

Construction of Recombinant HVT Expressing PmpD, and Immunological Evaluation against Chlamydia psittaci and Marek's Disease Virus.

Chlamydia psittaci (C. psittaci) is an obligate intracellular zoonotic pathogen that can be transmitted to humans from birds. No efficacious commercial vaccine is available for clearing chlamydial infection due to lack of potential vaccine candidates and effective delivery vehicles. Herpesvirus of turkeys (HVT) is an efficacious commercially available vaccine against Marek's Disease virus (MDV). In this study, a recombinant HVT-delivered vaccine against C. psittaci and Marek's disease was developed and examined. The 5'-terminus of pmpD gene (pmpD-N) encoding the N-terminal fragment of polymorphic membrane protein D of C. psittaci was inserted into a nonessential region of HVT genome using reverse genetics based on an infectious bacterial artificial chromosome (BAC) clone of HVT. The recombinant virus (rHVT-pmpD-N) was recovered from primary chicken embryo fibroblast (CEF) cells by transfection of modified HVT BAC DNA containing the pmpD-N gene. The rHVT-pmpD-N construct was confirmed to express PmpD-N by immunoblot and immunofluorescence. The rHVT-pmpD-N was stable during 20 passages in vitro. The growth kinetics of rHVT-pmpD-N was comparable to that of parental HVT in vitro and in vivo. One-day-old SPF chickens inoculated subcutaneously with rHVT-pmpD-N displayed increased PmpD-specific antibody levels and a vigorous PmpD-specific lymphocyte proliferation response using HVT vector or CEF cells as control. Furthermore, the percentage of CD4+ cells was significantly elevated in rHVT-pmpD-N-immunized birds as compared to the parental HVT. All chickens vaccinated with rHVT-pmpD-N or parental HVT were protected completely against challenge with a very virulent strain of Marek's Disease virus (MDV) RB-1B. Post challenge with C. psittaci CB7 strain, a significant decrease in respiratory distress, lesions and Chlamydia load was found in the rHVT-pmpD-N-vaccinated group compared to the parental HVT. In conclusion, our study suggests that the rHVT-pmpD-N live vaccine may be viable as a candidate dual vaccine that provides protection against both very virulent MDV and C. psittaci.

Risk exposures for human ornithosis in a poultry processing plant modified by use of personal protective equipment: an analytical outbreak study.

Ornithosis outbreaks in poultry processing plants are well-described, but evidence for preventive measures is currently lacking. This study describes a case-control study into an outbreak of ornithosis at a poultry processing plant in the East of England, identified following three employees being admitted to hospital. Workers at the affected plant were recruited via their employer, with exposures assessed using a self-completed questionnaire. Cases were ascertained using serological methods or direct antigen detection in sputum. 63/225 (28%) staff participated, with 10% of participants showing evidence of recent infection. Exposure to the killing/defeathering and automated evisceration areas, and contact with viscera or blood were the main risk factors for infection. Personal protective equipment (goggles and FFP3 masks) reduced the effect of exposure to risk areas and to self-contamination with potentially infectious material. Our study provides some evidence of effectiveness for respiratory protective equipment in poultry processing plants where there is a known and current risk of ornithosis. Further studies are required to confirm this tentative finding, but in the meantime respiratory protective equipment is recommended as a precautionary measure in plants where outbreaks of ornithosis occur.

Protective efficacy against Chlamydophila psittaci by oral immunization based on transgenic rice expressing MOMP in mice.

Avian chlamydiosis is caused by Chlamydophila psittaci (Cp. psittaci) and major outer membrane protein (MOMP) of Cp. psittaci is an excellent vaccine candidate. In this study, the MOMP gene was expressed in rice callus by the Agrobacterium tumefaciens vector. The production of protein in transgenic rice seeds was confirmed and quantified by Western-blot and ELISA, the results demonstrating that the antigen was expressed stably. The transgenic rice seeds expressing the MOMP protein were administered by the oral route to BALB/c mice, which developed MOMP-specific serum IgG and fecal IgA antibodies and a splenocyte MOMP-specific proliferative response and significant levels of IFN-γ, IL-2, IL-4, IL-5 and TGF-ß production. Immunization with MOMP transgenic seeds induced partial protection (50%) against a lethal challenge with the highly virulent Cp. psittaci 6BC strain. Lung function after challenge was less affected compared non-MOMP immunized animals. The results demonstrate the feasibility of using transgenic rice seeds as an oral vaccine to generate protective immunity and reduce the lung lesions in mice against virulent Cp. psittaci 6BC strain. This finding has implications for further development of an oral vaccine against avian chlamydiosis.

Risk assessment and management of Chlamydia psittaci in poultry processing plants.

Chlamydia psittaci causes respiratory disease in poultry and can be transmitted to humans. Historical outbreaks of psittacosis in poultry workers indicated the need for higher awareness and an efficient risk assessment and management. This group reviewed relevant previous research, practical guidelines, and European directives. Subsequently, basic suggestions were made on how to assess and manage the risk of psittacosis in poultry processing plants based on a classical four-step approach. Collective and personal protective measures as well as the role of occupational medicine are described. Despite the finding that exposure is found in every branch, abattoir workstations seem to be associated with the highest prevalence of psittacosis. Complete eradication is difficult to achieve. Ventilation, cleaning, hand hygiene, and personal protective equipment are the most important protective measures to limit and control exposure to C. psittaci. Adequate information, communication, and health surveillance belong to the responsibilities of the occupational physician. Future challenges lay in the rigorous reporting of infections in both poultry and poultry workers and in the development of an avian and human vaccine.

Vaccination of turkeys against Chlamydophila psittaci through optimised DNA formulation and administration.

We have demonstrated that vaccination of turkeys with an unformulated DNA vaccine induces significant protection against Chlamydophila (Cp.) psittaci infections. Nevertheless, the immunogenicity of the DNA vaccine can still be improved by increasing translation and transfection efficiency. Therefore, the ompA codon was adapted to the codon usage in birds, resulting in pcDNA1/MOMP(opt). To increase gene transfer, polyplexes of pcDNA1/MOMP(opt)-EGFP with different cationic polymers, such as linear and branched polyethyleneimine (lPEI and brPEI) and starburst PAMAM dendrimers, and lipoplexes with cationic DOTAP/DOPE liposomes were created. Transfection of lPEI and brPEI polyplexes with an N/P ratio of 8 resulted in the highest transfection efficiencies, but lPEI polyplexes were completely destroyed following nebulisation. Secondly, we examined the capacity of nebulised or intramuscularly (IM) administered brPEI-pcDNA1/MOMP(opt) to induce a significant protective immune response in SPF turkeys experimentally infected with 10(8) TCID(50) of a virulent Cp. psittaci strain. Results were compared to IM administration of naked plasmid DNA and to results of non-vaccinated animals. Intramuscular administration of brPEI-pcDNA1/MOMP(opt) increased the immunogenicity of the Cp. psittaci DNA vaccine as compared to IM administration of pcDNA1/MOMP(opt) or aerosol delivery of brPEI-pcDNA1/MOMP(opt). Improved immunogenicity was correlated with increased protection. Vaccinated groups were significantly protected against Cp. psittaci challenge.

Chlamydophila psittaci infections in turkeys: overview of economic and zoonotic importance and vaccine development.

We provide evidence on the multifactorial infectious etiology of respiratory disease in turkeys. Although Chlamydophila psittaci is difficult to diagnose, this entity should not be neglected in veterinary diagnostic laboratories. The present results suggest a pathogenic interplay between chlamydophila, avian metapneumovirus and Ornithobacterium rhinotracheale. Additionally, we demonstrate zoonotic transmission from turkeys to humans. Psittacosis due to contact with poultry probably occurs more often than is thought and the infection can be asymptomatic or symptomatic. There is no commercial C. psittaci vaccine available and currently the best option is an experimental major outer membrane protein-based DNA vaccine.

Protection of budgerigars (Melopsittacus undulatus) against Chlamydophila psittaci challenge by DNA vaccination.

Plasmid DNA (pcDNA1::MOMP A) expressing the major outer membrane protein (MOMP) of Chlamydophila psittaci genotype A strain 89/1051 has been tested for its ability to induce protective immunity against Cp. psittaci challenge in budgerigars. Eight pairs of male and female budgerigars were housed in eight separate bird cages placed in two negative pressure isolators, four cages per group. All budgerigars were immunised twice intramuscularly with 100 microasmid DNA. Both groups received a primary DNA inoculation at day 0 followed by a booster inoculation 3 weeks later. Group 1 received pcDNA1::MOMP A, while group 2 received the placebo vaccine pcDNA1. Budgerigars were challenged by aerosol 2 weeks following the booster vaccination. The challenge consisted of 10(8) TCID(50) of the homologous Cp. psittaci genotype A strain. Cloacal and pharyngeal swabs of all budgerigars, taken prior to the experimental infection were negative in both PCR and culture. However, all budgerigars showed low pre-existing serum antibody titres. This indicates that animals were previously infected. Nevertheless, DNA immunisation could significantly reduce clinical signs, macroscopic lesions, pharyngeal and cloacal excretion as well as chlamydial replication, even in the presence of pre-existing serum antibodies, as compared to the placebo-vaccinated controls.

Mucosal immunity in mice induced by orally administered transgenic rice.

Transgenic plants are efficient means of producing and delivering oral vaccines. Rice material shown previously to express the Chlamydophila psittaci (Cp. psittaci) antigen (MOMP) fused to the B subunit of Escherichia coli heat-labile enterotoxin (LTB) was fed to mice and the resulting immune response was investigated. Oral immunization of mice with the transgenic rice elicited MOMP-specific sera IgG and IgA antibodies, a strong increase of the lymphoproliferative response, and significant levels of IFN-gamma, TGF-beta and IL-2 production. Furthermore, the immunization of mice with transgenic rice elicited strong cytotoxic T lymphocyte (CTL) responses in vitro. These results demonstrated that plant-made LTB-MOMP fusion protein could induce significant humoral and cellular Th1 and Th3 immune responses. Moreover, transgenic rice immunization induced partial protection (53.3%) against a lethal challenge with the highly virulent Cp. psittaci 6BC strain in a BALB/c mouse model. These results suggest that expression of protective antigens of Cp. psittaci in transgenic rice has potential as an edible vaccine against avain chlamydiosis.

Evaluation of the prophylactic use of ovotransferrin against chlamydiosis in SPF turkeys.

Chlamydophila (C.) psittaci infections are highly prevalent in turkeys and the economical and public health importance of these infections has been recognized since 1950. As there are no vaccines, antibiotic treatment (tetracylines, enrofloxacine) is often needed to allow marketing of poultry. In this study, we explored the use of ovotransferrin (ovoTF), a natural anti-microbial protein, in preventing an experimental C. psittaci infection in specific pathogen free (SPF) turkeys. Turkeys were treated with aerosolized ovoTF prior to the infection. Groups 1 and 2 received a single dose of 10 and 5 mg ovoTF per turkey, respectively. Group 3 received a daily dose of 5mg ovoTF per turkey during 12 days. Group 4 served as untreated, infected control group. Turkeys were aerosol infected using 10(6) TCID(50) of the virulent C. psittaci serovar/genotype D strain 92/1293. Birds were monitored (clinical signs, bacterial excretion) during 12 subsequent days before being necropsied. At necropsy, pathology and C. psittaci replication in various tissues was examined. A single dose of 10mg ovoTF and a repeated daily dose of 5mg ovoTF could not prevent the birds from becoming infected with C. psittaci, but they significantly reduced the outcome of the infection. A single dose of 5mg ovoTF had no influence on the outcome of the infection as compared to the non-treated infected controls. Our results demonstrate the anti-chlamydial effect of ovoTF in vivo and present a base for further research on practical applications of ovoTF on turkey farms.

An outbreak of psittacosis in a bird park in Japan.

An outbreak of psittacosis related to a bird park occurred in Matsue City, Shimane Prefecture, Japan, during winter 2001. Seventeen cases of psittacosis (12 visitors, three staff, and two student interns) were confirmed. A cohort study was conducted among the park staff and students to determine the risk factors for the development of acute serologically confirmed psittacosis (SCP) infection. Being 'bird staff' had an increased risk of SCP infection (RR 3.96, 95% CI 1.48-10.58). Entering the staff building, where ill birds were maintained without proper isolation, was also associated with an increased risk of SCP infection (RR 3.61, 95% CI 1.03-12.6). Isolation of ill birds and quarantine measures were found to be insufficient. Dehumidifiers and a high-pressure water spray under a closed ventilation environment may have raised the concentration of Chlamydophila psittaci in the hothouses. Bird park staff and visitors should be educated about psittacosis.