A molecular epidemiological investigation of contagious caprine pleuropneumonia in goats and captive Arabian sand gazelle (Gazella marica) in Oman.

BACKGROUND: Contagious caprine pleuropneumonia (CCPP) is a fatal WOAH-listed, respiratory disease in small ruminants with goats as primary hosts that is caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp). Twelve CCPP outbreaks were investigated in 11 goat herds and a herd of captive Arabian sand gazelle (Gazella marica) in four Omani governorates by clinical pathological and molecular analysis to compare disease manifestation and Mccp genetic profiles in goats and wild ungulates. RESULTS: The CCPP forms in diseased and necropsied goats varied from peracute (5.8%), acute (79.2%) and chronic (4.5%) while all of the five necropsied gazelles showed the acute form based on the clinical picture, gross and histopathological evaluation. Colonies of Mccp were recovered from cultured pleural fluid, but not from lung tissue samples of one gazelle and nine goats and all the isolates were confirmed by Mccp-specific real time PCR. Whole genome-single nucleotide polymorphism (SNP) analysis was performed on the ten isolates sequenced in this study and twenty sequences retrieved from the Genbank database. The Mccp strains from Oman clustered all in phylogroup A together with strains from East Africa and one strain from Qatar. A low variability of around 125 SNPs was seen in the investigated Omani isolates from both goats and gazelles indicating mutual transmission of the pathogen between wildlife and goats. CONCLUSION: Recent outbreaks of CCPP in Northern Oman are caused by Mccp strains of the East African Phylogroup A which can infect goats and captive gazelles likewise. Therefore, wild and captive ungulates should be considered as reservoirs and included in CCPP surveillance measures.

De novo genome assembly resolving repetitive structures enables genomic analysis of 35 European Mycoplasmopsis bovis strains.

Mycoplasmopsis (M.) bovis, the agent of mastitis, pneumonia, and arthritis in cattle, harbors a small genome of approximately 1 Mbp. Combining data from Illumina and Nanopore technologies, we sequenced and assembled the genomes of 35 European strains and isolate DL422_88 from Cuba. While the high proportion of repetitive structures in M. bovis genomes represent a particular challenge, implementation of our own pipeline Mycovista (available on GitHub www.github.com/sandraTriebel/mycovista ) in a hybrid approach enabled contiguous assembly of the genomes and, consequently, improved annotation rates considerably. To put our European strain panel in a global context, we analyzed the new genome sequences together with 175 genome assemblies from public databases. Construction of a phylogenetic tree based on core genes of these 219 strains revealed a clustering pattern according to geographical origin, with European isolates positioned on clades 4 and 5. Genomic data allowing assignment of strains to tissue specificity or certain disease manifestations could not be identified. Seven strains isolated from cattle with systemic circular condition (SCC), still a largely unknown manifestation of M. bovis disease, were located on both clades 4 and 5. Pairwise association analysis revealed 108 genomic elements associated with a particular clade of the phylogenetic tree. Further analyzing these hits, 25 genes are functionally annotated and could be linked to a M. bovis protein, e.g. various proteases and nucleases, as well as ten variable surface lipoproteins (Vsps) and other surface proteins. These clade-specific genes could serve as useful markers in epidemiological and clinical surveys.

Looking Inside Non-Destructively: Label-Free, Raman-Based Visualization of Intracellular Coxiella burnetii.

The life cycle of intracellular pathogens is often complex and can include different morphoforms. Treatment of intracellular infections and unperturbed studying of the pathogen inside the host cell are frequently challenging. Here, we present a Raman-based, label-free, non-invasive, and non-destructive method to localize, visualize, and even quantify intracellular bacteria in 3D within intact host cells in a Coxiella burnetii infection model. C. burnetii is a zoonotic obligate intracellular pathogen that causes infections in ruminant livestock and humans with an acute disease known as Q fever. Using statistical data analysis, no isolation is necessary to gain detailed information on the intracellular pathogen's metabolic state. High-quality false color image stacks with diffraction-limited spatial resolution enable a 3D spatially resolved single host cell analysis that shows excellent agreement with results from transmission electron microscopy. Quantitative analysis at different time points post infection allows to follow the infection cycle with the transition from the large cell variant (LCV) to the small cell variant (SCV) at around day 6 and a gradual change in the lipid composition during vacuole maturation. Spectral characteristics of intracellular LCV and SCV reveal a higher lipid content of the metabolically active LCV.

Chronic wasting associated with Chlamydia pneumoniae in three ex situ breeding facilities for tropical frogs.

A number of different Chlamydia spp. have been detected in the class Amphibia with C. pneumoniae being the predominant species involved. Chlamydiae have been linked to mass mortality events, thereby representing significant pathogens that deserve attention with respect to worldwide amphibian decline. We here present six cases of chlamydiosis and asymptomatic chlamydial infections in different frog species from three ex situ amphibian conservation facilities. Clinical signs predominantly characterised by regurgitation, chronic wasting, lethargy and suspended breeding were associated with C. pneumoniae infection. Despite various treatment regimens, it was not possible to clear infections. However, intra vitam diagnostics succeeded from skin, faeces and urine for the first time.

Characterization of Biosynthetic Pathways for the Production of the Volatile Homoterpenes DMNT and TMTT in Zea mays.

Plant volatiles not only have multiple defense functions against herbivores, fungi, and bacteria, but also have been implicated in signaling within the plant and toward other organisms. Elucidating the function of individual plant volatiles will require more knowledge of their biosynthesis and regulation in response to external stimuli. By exploiting the variation of herbivore-induced volatiles among 26 maize (Zea mays) inbred lines, we conducted a nested association mapping and genome-wide association study (GWAS) to identify a set of quantitative trait loci (QTLs) for investigating the pathways of volatile terpene production. The most significant identified QTL affects the emission of (E)-nerolidol, linalool, and the two homoterpenes (E)-3,8-dimethyl-1,4,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). GWAS associated a single nucleotide polymorphism in the promoter of the gene encoding the terpene synthase TPS2 with this QTL Biochemical characterization of TPS2 verified that this plastid-localized enzyme forms linalool, (E)-nerolidol, and (E,E)-geranyllinalool. The subsequent conversion of (E)-nerolidol into DMNT maps to a P450 monooxygenase, CYP92C5, which is capable of converting nerolidol into DMNT by oxidative degradation. A QTL influencing TMTT accumulation corresponds to a similar monooxygenase, CYP92C6, which is specific for the conversion of (E,E)-geranyllinalool to TMTT The DMNT biosynthetic pathway and both monooxygenases are distinct from those previously characterized for DMNT and TMTT synthesis in Arabidopsis thaliana, suggesting independent evolution of these enzymatic activities.

Mycoplasma pneumoniae and Chlamydia spp. infection in community-acquired pneumonia, Germany, 2011-2012.

Mycoplasma pneumoniae and Chlamydia spp., which are associated with community-acquired pneumonia (CAP), are difficult to propagate, and can cause clinically indistinguishable disease patterns. During 2011-2012, we used molecular methods to test adult patients in Germany with confirmed CAP for infection with these 2 pathogens. Overall, 12.3% (96/783) of samples were positive for M. pneumoniae and 3.9% (31/794) were positive for Chlamydia spp.; C. psittaci (2.1%) was detected more frequently than C. pneumoniae (1.4%). M. pneumoniae P1 type 1 predominated, and levels of macrolide resistance were low (3.1%). Quarterly rates of M. pneumoniae-positive samples ranged from 1.5% to 27.3%, showing a strong epidemic peak for these infections, but of Chlamydia spp. detection was consistent throughout the year. M. pneumoniae-positive patients were younger and more frequently female, had fewer co-occurring conditions, and experienced milder disease than did patients who tested negative. Clinicians should be aware of the epidemiology of these pathogens in CAP.

In Vitro Antibacterial Activity of Microbial Natural Products against Bacterial Pathogens of Veterinary and Zoonotic Relevance.

Antimicrobial resistance (AMR) is considered one of the greatest threats to both human and animal health. Efforts to address AMR include implementing antimicrobial stewardship programs and introducing alternative treatment options. Nevertheless, effective treatment of infectious diseases caused by bacteria will still require the identification and development of new antimicrobial agents. Eight different natural products were tested for antimicrobial activity against seven pathogenic bacterial species (Brachyspira sp., Chlamydia sp., Clostridioides sp., Mannheimia sp., Mycobacterium sp., Mycoplasma sp., Pasteurella sp.). In a first pre-screening, most compounds (five out of eight) inhibited bacterial growth only at high concentrations, but three natural products (celastramycin A [CA], closthioamide [CT], maduranic acid [MA]) displayed activity at concentrations <2 µg/mL against Pasteurella sp. and two of them (CA and CT) also against Mannheimia sp. Those results were confirmed by testing a larger collection of isolates encompassing 64 Pasteurella and 56 Mannheimia field isolates originating from pigs or cattle, which yielded MIC90 values of 0.5, 0.5, and 2 µg/mL against Pasteurella and 0.5, 4, and >16 µg/mL against Mannheimia for CA, CT, and MA, respectively. CA, CT, and MA exhibited higher MIC50 and MIC90 values against Pasteurella isolates with a known AMR phenotype against commonly used therapeutic antimicrobial agents than against isolates with unknown AMR profiles. This study demonstrates the importance of whole-cell antibacterial screening of natural products to identify promising scaffolds with broad- or narrow-spectrum antimicrobial activity against important Gram-negative veterinary pathogens with zoonotic potential.

Mycoplasma mycoides subspecies capri, an uncommon mastitis and respiratory pathogen isolated in a German flock of goats.

Mycoplasma mycoides ssp. capri (Mmc) is one of the etiological microorganisms of contagious agalactia, which is among the diseases causing the highest economical losses in small ruminants. We report a disease outbreak in a German flock that led to significant suffering of goats characterized by mastitis, arthritis, pleuropneumonia and sudden deaths. Mmc was persistently isolated from many animals both from milk, and from a number of different swab and tissue samples. A number of closely related Mycoplasma spp. have to be taken into consideration to rule out important animal epizootics listed by European Animal Health Law and the World Organisation for Animal Health (WOAH). Some goats developed cross-reacting antibodies against Mycoplasma mycoides ssp. mycoides. Although Mmc is believed to be an uncommon microorganism in Germany, this study highlights that veterinarians should consider this pathogen in their work during herd health monitoring in Central Europe. Although eradication was not fully achieved, autogenous vaccination significantly seemed to improve animal health and welfare.

Localization of sesquiterpene formation and emission in maize leaves after herbivore damage.

BACKGROUND: Maize (Zea mays L.) leaves damaged by lepidopteran herbivores emit a complex volatile blend that can attract natural enemies of the herbivores and may also have roles in direct defense and inter- or intra-plant signaling. The volatile blend is dominated by sesquiterpenes of which the majority is produced by two herbivore-induced terpene synthases, TPS10 and TPS23. However, little is known about the pattern of volatile emission within maize leaves. RESULTS: In this study, we restricted herbivore feeding to small sections of the maize leaf with the aim of determining the patterns of volatile sesquiterpene emission throughout the damaged leaf and in neighboring leaves. Sesquiterpene volatiles were released at high rates from damaged leaves, but at much lower rates from neighboring leaves. Release was restricted to the site of damage or to leaf sections located apical to the damage, but was not seen in sections basal to the damage or on the other side of the midrib. The emission pattern correlated well with the transcript pattern of the respective sesquiterpene synthase genes, tps10 and tps23, implying that biosynthesis likely occurs at the site of emission. The concentrations of jasmonic acid and its leucine derivative were also elevated in terpene-emitting tissues suggesting a role for jasmonates in propagating the damage signal. CONCLUSIONS: In contrast to other defense reactions which often occur systemically throughout the whole plant, herbivore-induced sesquiterpene production in maize is restricted to the wounding site and distal leaf parts. Since the signal mediating this reaction is directed to the leaf tip and cannot propagate parallel to the leaf axis, it is likely connected to the xylem. The increasing gradient of volatiles from the tip of the leaf towards the damage site might aid herbivore enemies in host or prey finding.

Avian Chlamydia abortus Strains Detected in Galápagos Waved Albatross (Phoebastria irrorata).

Galápagos Penguin (Spheniscus mendiculus), Flightless Cormorant (Phalacrocorax harrisi), and Waved Albatross (Phoebastria irrorata) are among the most vulnerable species to natural and anthropogenic factors in the Galápagos Islands. In 2017, a dedicated study was conducted to detect Chlamydiaceae on cloacal swabs collected from 59 albatrosses, 68 penguins, and 10 cormorants in different islands and sites in the Galápagos Archipelago. A real-time PCR method targeting the conserved 23S ribosomal RNA gene of the Chlamydiaceae family detected the presence of the bacterium only in albatrosses from Punta Suárez, Española Island, with 21 positive samples (35.6%), whereas negative results were obtained with available real-time PCR systems specific to Chlamydia psittaci and Chlamydia abortus. Multilocus sequence typing (MLST) of the most strongly positive samples revealed a new sequence type closely related to the recently described avian strains of C. abortus. For a quick identification, a new real-time PCR system that allows the detection of all strains (avian and ruminant) belonging to the C. abortus species has been developed. Applied to a second set of samples from 31 albatrosses collected at Punta Suárez, Española Island, in 2018, the new real-time PCR system confirmed the presence of this bacteria in this group of birds, with the same new MLST sequence type.

Whole Genome Sequencing and Comparative Genomic Analysis of Chlamydia gallinacea Field Strains Isolated from Poultry in Poland.

Chlamydia gallinacea is an intracellular bacterium belonging to the Chlamydiaceae family. Poultry is considered to be the major reservoir of this agent, which has worldwide distribution and a particularly consistent worldwide occurrence in chicken flocks. The bacterium has been linked to respiratory disease in humans but without definitive confirmation; nevertheless, while it has not been proved to be the cause of human respiratory disease, a recent report from Italy verified its bird-to-human transmission. This aspect being significant for public health, more research is needed to gain insight into the infection biology of C. gallinacea. In this study, the genomes of eleven novel C. gallinacea field strains from different regions of Poland were analyzed comparatively. It was confirmed that C. gallinacea strains are closely related, with at least 99.46% sequence identity. They possess a conservative genome structure involving the plasticity zone with a complete cytotoxin, the type three secretion system, inclusion membrane proteins, polymorphic membrane proteins, hctA and hctB histone-like proteins, and the chlamydial protease-like activating factor exoenzyme, as well as plasmids. Genetic diversity seems to be restricted. However, some genetic loci, such as ompA and multi-locus sequence typing target genes, are diverse enough to enable high-resolution genotyping and epidemiological tracing.

A new duplex qPCR-based method to quantify Mycoplasma mycoides in complex cell culture systems and host tissues.

Bacterial pathogen-host interactions are a complex process starting with adherence and colonization followed by a variety of interactions such as invasion or cytotoxicity on one hand and pathogen recognition, secretion of proinflammatory/antibacterial substances and enhancing the barrier function of epithelial layers on the other hand. Therefore, a variety of in vitro, ex vivo and in vivo models have been established to investigate these interactions. Some in vitro models are composed of different cell types and extracellular matrices such as tissue explants or precision cut lung slices. These complex in vitro models mimic the in vivo situation more realistically, however, they often require new and more sophisticated methods for quantification of experimental results. Here we describe a multiplex qPCR-based method to quantify the number of bacteria of Mycoplasma (M.) mycoides interacting with their hosts in an absolute manner as well as normalized to the number of host cells. We choose the adenylate kinase (adk) gene from the pathogen and the Carcinoembryonic antigen-related cell adhesion molecule 18 (CEACAM18) gene from the host to determine cell numbers by a TaqMan-based assay system. Absolute copy numbers of the genes are calculated according to a standard containing a defined number of plasmids containing the sequence which is amplified by the qPCR. The new multiplex qPCR therefore allows the quantification of M. mycoides interacting with host cells in suspension, monolayer, 3D cell culture systems as well as in host tissues.

Occurrence of Chlamydiae in Corvids in Northeast Italy.

Chlamydiaceae occurrence has been largely evaluated in wildlife, showing that wild birds are efficient reservoirs for avian chlamydiosis. In this study, DNA extracted from cloacal swabs of 108 corvids from Northeast Italy was screened for Chlamydiaceae by 23S real-time (rt)PCR. The positive samples were characterised by specific rtPCRs for Chlamydia psittaci, Chlamydia abortus, Chlamydia gallinacea, Chlamydia avium, Chlamydia pecorum and Chlamydia suis. Cloacal shedding of Chlamydiaceae was detected in 12 out of 108 (11.1%, 5.9%-18.6% 95% CI) corvids sampled. Molecular characterisation at the species level was possible in 8/12 samples, showing C. psittaci positivity in only one sample from a hooded crow and C. abortus positivity in seven samples, two from Eurasian magpies and five from hooded crows. Genotyping of the C. psittaci-positive sample was undertaken via PCR/high-resolution melting, clustering it in group III_pigeon, corresponding to the B genotype based on former ompA analysis. For C. abortus genotyping, multilocus sequence typing was successfully performed on the two samples with high DNA load from Eurasian magpies, highlighting 100% identity with the recently reported Polish avian C. abortus genotype 1V strain 15-58d44. To confirm the intermediate characteristics between C. psittaci and C. abortus, both samples, as well as two samples from hooded crows, showed the chlamydial plasmid inherent in most C. psittaci and avian C. abortus, but not in ruminant C. abortus strains. The plasmid sequences were highly similar (≥99%) to those of the Polish avian C. abortus genotype 1V strain 15-58d44. To our knowledge, this is the first report of avian C. abortus strains in Italy, specifically genotype 1V, confirming that they are actively circulating in corvids in the Italian region tested.

Assessment of a novel multiplex real-time PCR assay for the detection of the CBPP agent Mycoplasma mycoides subsp. mycoides SC through experimental infection in cattle.

BACKGROUND: Mycoplasma mycoides subsp. mycoides SC is the pathogenic agent of contagious bovine pleuropneumonia (CBPP), the most important disease of cattle in Africa causing significant economic losses. The re-emergence of CBPP in Europe in the 1980s and 1990s illustrates that it is still a threat also to countries that have successfully eradicated the disease in the past. Nowadays, probe-based real-time PCR techniques are among the most advanced tools for a reliable identification and a sensitive detection of many pathogens, but only few protocols have been published so far for CBPP diagnosis. Therefore we developed a novel TaqMan®-based real-time PCR assay comprising the amplification of two independent targets (MSC_0136 and MSC_1046) and an internal exogenous amplification control in a multiplex reaction and evaluated its diagnostic performance with clinical samples. RESULTS: The assays detected 49 MmmSC strains from diverse temporal and geographical origin, but did not amplify DNA from 82 isolates of 20 non-target species confirming a specificity of 100%. The detection limit was determined to be 10 fg DNA per reaction for the MSC_0136 assay and 100 fg per reaction for the MSC_1046 assay corresponding to 8 and 80 genome equivalents, respectively. The diagnostic performance of the assay was evaluated with clinical samples from 19 experimentally infected cattle and from 20 cattle without CBPP and compared to those of cultivation and a conventional PCR protocol. The two rt-PCR tests proved to be the most sensitive methods and identified all 19 infected animals. The different sample types used were not equally suitable for MmmSC detection. While 94.7% of lung samples from the infected cohort were positively tested in the MSC_0136 assay, only 81% of pulmonal lymph nodes, 31% of mediastinal lymph nodes and 25% of pleural fluid samples gave a positive result. CONCLUSIONS: The developed multiplex rt-PCR assay is recommended as an efficient tool for rapid confirmation of a presumptive CBPP diagnosis in a well-equipped laboratory environment.

Pmp Repertoires Influence the Different Infectious Potential of Avian and Mammalian Chlamydia psittaci Strains.

Chlamydia psittaci is the etiological agent of chlamydiosis in birds and can be transmitted to humans, causing severe systemic disease. C. psittaci infects a broad range of hosts; strains are isolated not only from birds but also from mammals, where they seem to have a reduced infectious and zoonotic potential. Comparative analysis of chlamydial genomes revealed the coding sequences of polymorphic membrane proteins (Pmps) to be highly variable regions. Pmps are characterized as adhesins in C. trachomatis and C. pneumoniae and are immunoreactive proteins in several Chlamydia species. Thus, Pmps are considered to be associated with tissue tropism and pathogenicity. C. psittaci harbors 21 Pmps. We hypothesize that the different infectious potential and host tropism of avian and mammalian C. psittaci strains is dependent on differences in their Pmp repertoires. In this study, we experimentally confirmed the different virulence of avian and mammalian strains, by testing the survival rate of infected embryonated eggs and chlamydiae dissemination in the embryos. Further, we investigated the possible involvement of Pmps in host tropism. Analysis of pmp sequences from 10 C. psittaci strains confirmed a high degree of variation, but no correlation with host tropism was identified. However, comparison of Pmp expression profiles from different strains showed that Pmps of the G group are the most variably expressed, also among avian and mammalian strains. To investigate their functions, selected Pmps were recombinantly produced from one avian and one mammalian representative strain and their adhesion abilities and relevance for the infection of C. psittaci strains in avian and mammalian cells were tested. For the first time, we identified Pmp22D, Pmp8G, and OmcB as relevant adhesins, essential during infection of C. psittaci strains in general. Moreover, we propose Pmp17G as a possible key player for host adaptation, as it could only bind to and influence the infection in avian cells, but it had no relevant impact towards infection in mammalian cells. These data support the hypothesis that distinct Pmp repertoires in combination with specific host factors may contribute to host tropism of C. psittaci strains.

Development of a species-specific real-time PCR test for Chlamydia psittaci and its employment in the investigation of zoonotic transmission from racing pigeons in Denmark.

Species-specific detection of Chlamydia psittaci is challenging and all published PCR tests have so far shown deficiencies in specificity or sensitivity. The present investigation reports on the development of a species-specific real-time PCR assay for C. psittaci. The test is based on an 84 bp indel in a gene of unknown function that is unique to C. psittaci. The Cps-indel84-PCR assay was validated on a wide range of chlamydial and other bacterial strains as well as on clinical samples from animals and humans in two different diagnostic laboratories in Germany and Denmark. Furthermore, the test was employed for investigating samples from racing pigeon flocks in Denmark. The evaluation showed that the Cps-indel84-PCR assay has excellent test characteristics and is a highly reliable method for identifying C. psittaci in clinical samples both from humans and animals.

A New SNP-Based Genotyping Method for C. psittaci: Application to Field Samples for Quick Identification.

Chlamydia (C.) psittaci is the causative agent of avian chlamydiosis and human psittacosis. In this study, we extracted single-nucleotide polymorphisms (SNPs) from the whole genome sequences of 55 C. psittaci strains and identified eight major lineages, most of which are host-related. A combined PCR/high-resolution melting (HRM) assay was developed to screen for eight phylogenetically informative SNPs related to the identified C. psittaci lineages. The PCR-HRM method was validated on 11 available reference strains and with a set of 118 field isolates. Overall, PCR-HRM clustering was consistent with previous genotyping data obtained by ompA and/or MLST analysis. The method was then applied to 28 C. psittaci-positive samples from animal or human cases. As expected, PCR-HRM typing results from human samples identified genotypes linked to ducks and pigeons, a common source of human exposure, but also to the poorly described Mat116-like genotype. The new genotyping method does not require time-consuming sequencing and allows a quick identification of the source of infection.

Bovine Abortions Revisited-Enhancing Abortion Diagnostics by 16S rDNA Amplicon Sequencing and Fluorescence in situ Hybridization.

Abortion in cattle causes significant economic losses for cattle farmers worldwide. The diversity of abortifacients makes abortion diagnostics a complex and challenging discipline that additionally is restrained by time and economy. Microbial culture has traditionally been an important method for the identification of bacterial and mycotic abortifacients. However, it comes with the inherent bias of favoring the easy-to-culture species, e.g., those that do not require cell culture, pre-enrichment, a variety of selective growth media, or different oxygen levels for in vitro growth. Molecular methods such as polymerase chain reaction (PCR) and next-generation sequencing have been established as alternatives to traditional microbial culturing methods in several diagnostic fields including abortion diagnostics. Fluorescence in situ hybridization (FISH), a bridging microscopy technique that combines molecular accuracy with culture independence, and spatial resolution of the pathogen-lesion relation, is also gaining influence in several diagnostic fields. In this study, real-time quantitative PCR (qPCR), 16S rDNA amplicon sequencing, and FISH were applied separately and in combination in order to (i) identify potentially abortifacient bacteria without the bias of culturability, (ii) increase the diagnostic rate using combined molecular methods, (iii) investigate the presence of the difficult-to-culture zoonotic agents Coxiella burnetii, Chlamydia spp., and Leptospira spp. in bovine abortions in Denmark. Tissues from 162 aborted or stillborn bovine fetuses and placentas submitted for routine diagnostics were screened for pathogenic bacteria using 16S rDNA amplicon sequencing. Lesion association of fungal elements, as well as of selection of bacterial abortifacients, was assessed using specific FISH assays. The presence of Chlamydia spp. and chlamydia-like organisms was assessed using qPCR. The study focused on bacterial and fungal abortifacients, because Danish cattle is free from most viral abortifacients. The 16S rDNA amplicon sequencing-guided FISH approach was suitable for enhancing abortion diagnostics, i.e., the diagnostic rate for cases with tissue lesions (n = 115) was increased from 46 to 53% when compared to routine diagnostic methods. Identification of Bacillus licheniformis, Escherichia coli, and Trueperella pyogenes accounted for the majority of additional cases with an established etiology. No evidence for emerging or epizootic bacterial pathogens was found. The difficult-to-culture abortifacients were either not detected or not identified as abortifacients.

Development of a Plasmid Shuttle Vector System for Genetic Manipulation of Chlamydia psittaci.

The obligate intracellular bacterium Chlamydia psittaci is a known avian pathogen causing psittacosis in birds and is capable of zoonotic transmission. In human pulmonary infections, C. psittaci can cause pneumonia associated with significant mortality if inadequately diagnosed and treated. Although intracellular C. psittaci manipulates host cell organelles for its replication and survival, it has been difficult to demonstrate host-pathogen interactions in C. psittaci infection due to the lack of easy-to-handle genetic manipulation tools. Here, we show the genetic transformation of C. psittaci using a plasmid shuttle vector that contains a controllable gene induction system. The 7,553-bp plasmid p01DC12 was prepared from the nonavian C. psittaci strain 01DC12. We constructed the shuttle vector pCps-Tet-mCherry using the full sequence of p01DC12 and the 4,449-bp fragment of Chlamydia trachomatis shuttle vector pBOMB4-Tet-mCherry. pCps-Tet-mCherry includes genes encoding the green fluorescent protein (GFP), mCherry, and ampicillin resistance (AmpR). Target genes can be inserted at a multiple cloning site (MCS). Importantly, these genes can be regulated by a tetracycline-inducible (tet) promoter. Using the pCps-Tet-mCherry plasmid shuttle vector, we show the expression of GFP, as well as the induction of mCherry expression, in C. psittaci strain 02DC15, which belongs to the avian C. psittaci 6BC clade. Furthermore, we demonstrated that pCps-Tet-mCherry was stably retained in C. psittaci transformants. Thus, our C. psittaci plasmid shuttle vector system represents a novel targeted approach that enables the elucidation of host-pathogen interactions.IMPORTANCE Psittacosis, caused by avian C. psittaci, has a major economic impact in the poultry industry worldwide and represents a significant risk for zoonotic transmission to humans. In the past decade, the tools of genetic manipulation have been improved for chlamydial molecular studies. While several genetic tools have been mainly developed in Chlamydia trachomatis, a stable gene-inducible shuttle vector system has not to date been available for C. psittaci In this study, we adapted a C. trachomatis plasmid shuttle vector system to C. psittaci We constructed a C. psittaci plasmid backbone shuttle vector called pCps-Tet-mCherry. The construct expresses GFP in C. psittaci Importantly, exogeneous genes can be inserted at an MCS and are regulated by a tet promoter. The application of the pCps-Tet-mCherry shuttle vector system enables a promising new approach to investigate unknown gene functions of this pathogen.

Comparative Genome Analysis of 33 Chlamydia Strains Reveals Characteristic Features of Chlamydia Psittaci and Closely Related Species.

To identify genome-based features characteristic of the avian and human pathogen Chlamydia(C.) psittaci and related chlamydiae, we analyzed whole-genome sequences of 33 strains belonging to 12 species. Using a novel genome analysis tool termed Roary ILP Bacterial Annotation Pipeline (RIBAP), this panel of strains was shown to share a large core genome comprising 784 genes and representing approximately 80% of individual genomes. Analyzing the most variable genomic sites, we identified a set of features of C. psittaci that in its entirety is characteristic of this species: (i) a relatively short plasticity zone of less than 30,000 nt without a tryptophan operon (also in C. abortus, C. avium, C. gallinacea, C. pneumoniae), (ii) a characteristic set of of Inc proteins comprising IncA, B, C, V, X, Y (with homologs in C. abortus, C. caviae and C. felis as closest relatives), (iii) a 502-aa SinC protein, the largest among Chlamydia spp., and (iv) an elevated number of Pmp proteins of subtype G (14 in C. psittaci, 14 in Cand. C. ibidis). In combination with future functional studies, the common and distinctive criteria revealed in this study provide important clues for understanding the complexity of host-specific behavior of individual Chlamydia spp.