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This paper provides an overview of the current knowledge of chlamydiae. These intracellular microorganisms belonging to the Chlamydiaceae family are widely distributed throughout the world. Constant development of culture-independent approaches for characterisation of microbial genomes enables new discoveries in the field of Chlamydia. The number of new taxa is continuously increasing as well as the range of hosts. New species and genotypes are constantly being discovered, particularly new avian and reptilian agents, which are discussed in this article. Interestingly, wild animals are the main hosts for new Chlamydia species including different species of bird, turtle and snake. The availability of next-generation sequencing opens up a new prospect for research and leads to deeper knowledge of these interesting microorganisms about which much is still to discover.

BackgroundCoxiella burnetiiis the etiological agent of Q fever, a zoonosis affecting many animal species including sheep and goats. The aims of this study were to evaluate the shedding ofCoxiella burnetiiin small ruminant herds and to identify the pathogen’s genotypes and sequence types (STs) using multiple-locus variable number tandem repeat analysis (MLVA) and multispacer sequence typing (MST) methods.ResultsOverall, 165 samples from 43 herds of goats and 9 flocks of sheep were collected including bulk tank milk (BTM), individual milk samples, vaginal swabs, tissue sections from stillborn kids, feces and placentas. These were tested by real-time PCR targeting the IS1111element.C. burnetiiinfection was confirmed in 51.16% of the herds of goats and 22.2% of the flocks of sheep. Six out of nine samples originating from goats were successfully genotyped using the MLVA method. The presence was confirmed of two widely distributed MLVA genotypes (I and J) and genotype PL1 previously reported only in cattle. Only one sequence type (ST61) was identified; however, the majority of specimens represented partial STs and some of them may belong to ST61. Other partial STs could possibly be ST74.ConclusionThis study confirmed the relatively common occurrence ofCoxiella burnetiiin small ruminant herds in Poland. Interestingly, all genotyped samples represent cattle-associated MLVA genotypes.

Introduction Q fever (coxiellosis) is an infectious disease of animals and humans, caused by.C. burnetii and widely distributed throughout the world. It is known that people and animals acquire the disease predominantly.via inhalation of infectious aerosols. The possibility of transmission of the pathogen by the alimentary route is still a matter of debate and remains controversial. Therefore the aim of this study was to fill the gaps in knowledge of oral transmission of.C. burnetii by conducting biological tests on the guinea pig model. Material and Methods Guinea pigs, divided into five groups comprising a negative control and four experimental groups, received specified concentrations of.C. burnetii per os. To determine the presence of specific antibodies, blood samples were tested using CFT. Also, internal organs collected during necropsy were screened by a real-time PCR targeting I.1111. Additionally, histopathological evaluation of the tissues was performed. Results The presence of antibodies and pathogen DNA in caecum was confirmed in one guinea pig from experimental group IV..C. burnetii was also detected in testicular tissue collected from one animal of experimental group II. Conclusions The presence of pathogen DNA in the testicular tissue indicates that infection spreads haematogenously. In the majority of experimental animals specific antibodies and genetic material of.C. burnetii were not detected. This fact suggests that development of infection depends on many factors, such as animal immune status.

Introduction: Coxiella (C.) burnetii, the aetiological agent of Q fever, is able to modulate the macrophage/T-lymphocyte axis in an infected organism and impair synthesis of monokines and lymphokines. Material and Methods: The purpose of this research was to determine the levels of the cytokines that play a key role in the response to C. burnetii antigens (IL-1β, IL-2, IL-6, IL-10, IFN-γ and TNF-α) in the serum of animals originating from an infected herd prior to vaccination (day 0) and at 1, 7, and 21 days afterwards. Results: The vaccination of animals did not affect the production of IL-6, IL-1β, or IL-2. The serum levels of these cytokines were too low to measure in most of the samples. The initial levels of TNFα, IFNγ, and IL-10 were higher in seropositive than in seronegative animals, although significant differences between seropositive shedders and seropositive nonshedders appeared only in the levels of IFNγ and IL-10. Additionally, the course of the post-vaccination response concerning these two cytokines was different among seronegative nonshedders, seropositive nonshedders, and seropositive shedders. Conclusion: It seems that analysis of the IFNγ and IL-10 concentrations in animal blood serum may have some practical value in an assessment of the health status of seropositive animals and post-vaccination response.

Here, we report the draft genome sequences of avian Chlamydia abortus genotype G2 strain 15-49d3, isolated from mallard, and genotype 1V strain 15-58d44, isolated from magpie in Poland. The total genome assembly lengths are 1,140,139 bp and 1,158,207 bp, respectively.

The aim of our study was the collection of seroprevalence data for Toxoplasma gondii, Coxiella burnetii, Trichinella spp., and Francisella tularensis from hunters in Lublin Province. The antibodies against T. gondii and C. burnetii were recorded in 38.5% and 16.2% of the sera, respectively. 4.05% of the sera were seropositive for both T. gondii and C. burnetii. None of the sera tested reacted positively with F. tulariensis or Trichinella spp. Seroprevalence of T. gondii and C. burnetii is common among the hunters from Lublin Province. It seems reasonable to undertake similar research among hunters from other regions of eastern Poland.

Introduction: The study was conducted to investigate the prevalence and genetic diversity of Chlamydia spp. in poultry in Poland and estimate possible transmission to humans. Material and Methods: Molecular diagnostic methods followed by sequencing and strain isolation were used on cloacal/faecal swabs collected from 182 apparently healthy poultry flocks including chickens, turkeys, ducks, and geese. Serum samples obtained from people exposed (study group) and non-exposed (control group) to birds were tested by complement fixation test to acquire data on Chlamydia spp. antibody level. Results: Overall, 15.9% of the tested flocks were Chlamydiaceae-positive and three Chlamydia spp. were identified. Predominant chlamydial agent found was C. gallinacea occurring in 65.5% of all positive poultry flocks and in 73.0% of positive chicken flocks. The sequences from four chicken flocks were assigned to C. abortus, whereas C. psittaci was confirmed in one duck and one goose flock. The analysis of ompA variable domains revealed at least nine genetic variants of C. gallinacea. Chlamydial antibodies were detected in 19.2% of human serum samples in the study group in comparison with 10.8% in the controls. Conclusion: The obtained results confirm that chlamydiae are common among chicken flocks in Poland with C. gallinacea as a dominant species. Moreover, the presence of C. abortus in chickens is reported here for the first time. Further investigation should focus on possible zoonotic transmission of C. gallinacea and C. abortus as well as potential pathogenic effects on birds’ health and poultry production.

A variety of Chlamydia species belonging to the Chlamydiaceae family have been reported in birds. Until recently, C. psittaci was considered to be the most common avian species, although found in both birds and mammals, while C. abortus has only been found in mammals. Recently, a new group of avian C. abortus strains with worldwide distribution in various wild bird families has been described. In this study, whole genome sequencing (WGS) of three of these strains (15-70d24, 15-49d3 and 15-58d44, representing genotypes G1, G2 and 1V, respectively) that were isolated from wild birds were analysed. Genome assemblies based on both short-read Illumina and long-read Nanopore data indicate that these avian C. abortus strains show features characteristic of both C. abortus and C. psittaci species, although phylogenetic analyses demonstrate a closer relationship with classical C. abortus strains. Currently, species classification established by the ICSP Subcommittee on the taxonomy of Chlamydiae, determines that these avian C. abortus strains 15-70d24, 15-49d3 and 15-58d44 should be classified as C. abortus. However, the authors of this study conclude that the current taxonomic definition of C. abortus is outdated and should be amended to include two subgroups, mammalian and avian, the latter of which would include all isolates so far referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates.

Introduction: Mycoplasma bovis is one of the main pathogens involved in cattle pneumonia. Other mycoplasmas have also been directly implicated in respiratory diseases in cattle. The prevalence of different Mycoplasma spp. in cattle affected by respiratory diseases and molecular characteristics of M. bovis field strains were evaluated. Material and Methods: In total, 713 nasal swabs from 73 cattle herds were tested. The uvrC gene fragment was amplified by PCR and PCR products were sequenced. PCR/DGGE and RAPD were performed. Results: It was found that 39 (5.5%) samples were positive for M. bovis in the PCR and six field strains had point nucleotide mutations. Additionally, the phylogenetic analysis of 20 M. bovis field strains tested with RAPD showed two distinct groups of M. bovis strains sharing only 3.8% similarity. PCR/DGGE analysis demonstrated the presence of bacteria belonging to the Mollicutes class in 79.1% of DNA isolates. The isolates were identified as: Mycoplasma bovirhinis, M. dispar, M. bovis, M. canis, M. arginini, M. canadense, M. bovoculi, M. alkalescens, and Ureaplasma diversum. Conclusion: Different Mycoplasma spp. strains play a crucial role in inducing respiratory diseases in cattle.

The spur-thighed tortoise (Testudo graeca) is an endangered Mediterranean tortoise that lives in North Africa, Southern Europe and Southwest Asia. In the wake of recent legislation making their keeping as domestic animals illegal, many of these animals have been returned to wildlife recovery centers in Spain. In the present study, a population of such tortoises showing signs of ocular disease and nasal discharge was examined for the presence of Chlamydia spp. Cloacal, conjunctival and/or choanal swabs were collected from 58 animals. Using a real-time PCR specific for the family Chlamydiaceae, 57/58 animals tested positive in at least one sample. While only a few samples proved positive for C. pecorum, sequencing of the 16S rRNA gene revealed a sequence identical to previously published sequences from specimens of German and Polish tortoises. Whole-genome sequences obtained from two conjunctival swab samples, as well as ANIb, TETRA values and a scheme based on 9 taxonomic marker genes revealed that the strain present in the Spanish tortoises represented a new yet non-classified species, with C. pecorum being its closest relative. We propose to designate the new species Candidatus Chlamydia testudinis.

Here, we report the draft genome sequence of avian Chlamydia abortus genotype G1 strain 15-70d24, isolated from Eurasian teal in Poland. The total genome assembly length is 1,149,382 bp.

Q fever is a worldwide zoonotic disease reported in humans and many animal species including cattle. The aims of this study were to evaluate the prevalence of Coxiella (C.) burnetii shedding in Polish dairy cattle herds and to identify the pathogen's genotypes and sequence types (STs) using multiple-locus variable number tandem repeat analysis (MLVA) and multispacer sequence typing (MST) methods. The presence of C. burnetii DNA was detected using a commercial real-time PCR kit, targeting the IS1111 element. Overall, 1,439 samples from 279 herds were tested including: 897 individual milk specimens, 101 bulk tank milk samples, 409 genital tract swabs and 32 placentas. Furthermore, 30 consumer milk samples, including 10 from vending machines and 77 dairy products were also analyzed. C. burnetii shedding was confirmed in 31.54% of tested cattle herds as well as in 69.16% of consumer milk and dairy products. Among real-time PCR-positive samples, 49 specimens obtained from 49 cattle herds and 8 samples of purchased dairy products were selected for genotyping. Overall, five previously known MLVA genotypes (I, J, BG, BE, and NM) and three new ones (proposed as PL1, PL2, and PL3) were identified. Two MST sequence types were recorded: ST16 and a novel sequence (ST61). The new genotypes and sequence types need further research particularly into their pathogenicity to humans.

The aim of the study was to assess the seroprevalence of Coxiella burnetii in cattle herds in different regions of Poland. A total of 1150 serum samples collected from 443 cattle herds from 14 provinces were tested using complement fixation test. The seroprevalence was different in individual regions of Poland. The average percentage of seropositive herds was 40.41% and these herds were identified in each province tested.

The aim of the research was to assess the prevalence of antibodies to Coxiella burnetii in dairy cattle herds in Poland and to compare the results of real-time PCR and ELISA tests performed on bulk tank milk (BTM) samples. In total, 2635 serum samples collected from 969 dairy cattle herds from all provinces were tested using ELISA. Additionally, BTM specimens from 101 herds were analysed by ELISA and real-time PCR targeting IS1111 element. Presence of anti-C. burnetii antibodies was confirmed in 25.39% of serum samples in 237 herds (24.46%) and the herd-level seroprevalence in Voivodeships varied from 2.5% to 61.4%. Moreover, 46 (45.5%) of analysed bulk tank milk samples gave postive result in ELISA and microbial DNA was detected in 40 (39.6%) of tested herds. The comparative analysis of ELISA and real-time PCR results obtained for BTM samples using the chi-square test showed statistically significant relationship between results of both methods.

Chlamydia (C.) abortus, a globally distributed obligate intracellular bacterium, has attracted increasing interest according to its veterinary importance and zoonotic nature. C. abortus can infect a variety of animals and cause foetal loss in livestock resulting in economic loss. In this study, the samples collected from two farms of foxes (n=20), raccoon dogs (n=15) and minks (n=20), were investigated by Chlamydiaceae- and Chlamydia species-specific real-time PCR. The results showed that all the tested foxes (20/20) and raccoon dogs (15/15) harbored Chlamydia spp., while 5% of minks (1/20) were positive for Chlamydia spp. C. abortus was identified in all positive samples as the dominant Chlamydia species, with C. pecorum DNA coexistence in some of the rectal samples (7/20) taken from foxes. Phylogenetic analysis based on specific gene fragments of 16S rRNA, IGS-23S rRNA, and ompA revealed that all sequences obtained in this study were assigned to the Chlamydiaceae family with high similarity to C. abortus S26/3 and B577 previously identified in ruminants. This is the first report confirming that farmed foxes, raccoon dogs, and minks carry C. abortus. Further studies are needed to fully elucidate the epidemiology and pathogenicity of this pathogen in farmed fur animals as well as the potential risks to public health.

Chlamydiales, one of the oldest bacterial orders in evolutionary terms, are widespread among animals. Blinding trachoma, a disease caused by Chlamydia trachomatis, was already known in ancient times, whereas modern reports on psittacosis date from 1879. Though these pathogens have long been known and lead to serious health problems both in human and animals, data on Chlamydiales biology has been limited. It is due to their intracellular life style and complex developmental cycle. New molecular biological methods have been recently developed expanding the possibilities of chlamydial research and diagnosis. This paper reviews data concerning avian chlamydiosis, its aetiological agent C. psittaci, newly proposed species isolated from birds, namely C. ibidis sp. nov., C. avium sp. nov., and C. gallinacea sp. nov., and their zoonotic potential.

This review presents recent data about avian chlamydiosis. Chlamydia psittaci has been considered to be the main causative agent of chlamydiosis in birds; however, two new Chlamydia species have been detected recently-C. gallinacea in breeding birds and C. avium in wild birds. We discuss the zoonotic potential of avian Chlamydia species.

Coxiella burnetii is the causative agent of Q fever. The cases of the disease are recorded in various species, including domestic animals. The aim of this investigation was to estimate the seroprevalence of C. burnetii in ruminants including cattle, sheep, goats, and horses. Totally, 2082 serum samples from 936 goats, 933 cattle, 89 sheep, and 124 horses, including various horse breeds, were tested by ELISA or complement fixation test. The examination revealed that Polish horses are seronegative while in the populations of cattle and small ruminants, seropositive animals are presented. The percentage of seropositive cattle, goats and sheep was 4.18, 6.30, and 13.48, respectively.

A variety of Chlamydia species belonging to the Chlamydiaceae family have been reported in reptilian hosts but scarce data about their occurrence in turtles and tortoises are available. In this study, research was conducted to acquire information on invasive alien species (IAS) of turtles and indigenous turtles and tortoises, living both free and in captivity, as possible reservoirs of Chlamydiaceae. Analysis of specimens (pharyngeal and cloacal swabs and tissues) from 204 turtles and tortoises revealed an overall Chlamydiaceae prevalence of 18.3% and 28.6% among free-living and captive animals respectively, with variable levels of shedding. Further testing conducted with a species-specific real-time PCR and microarray test was unsuccessful. Subsequently sequencing was applied to genotype the Chlamydiaceae-positive samples. Almost the full lengths of the 16S rRNA and ompA genes as well as the 16S-23S intergenic spacer (IGS) and 23S rRNA domain I were obtained for 14, 20 and 8 specimens respectively. Phylogenetic analysis of 16S rRNA amplicons revealed two distinct branches. Group 1 (10 specimens), specific to freshwater turtles and reported here for the first time, was most closely related to Chlamydia (C.) pneumoniae strains and the newly described Candidatus C. sanzinia. Group 2 (four specimens), detected in Testudo spp. samples, showed highest homology to C. pecorum strains but formed a separate sub-branch. Finally, molecular analysis conducted on positive samples together with their geographical distribution in places distant from each other strongly suggest that Group 1 specimens correspond to a new species in the Chlamydiaceae family. In-depth studies of Chlamydia spp. from turtles and tortoises are needed to further characterise these atypical strains and address arising questions about their pathogenicity and zoonotic potential.

Q fever is a zoonotic disease caused by Coxiella burnetii. The main source of infection are ruminants (cattle, sheep, and goats). C. burnetii is excreted via birth products, vaginal mucus, milk, and faeces. Raw milk is considered useful for epidemiological examinations of animals and evaluation of infection dynamics at the herd level. This article summarises data on prevalence studies on C. burnetii in bulk-tank milk in different European countries with the means of serological tests and PCR. It also summarises the results of studies to evaluate the actual risk of disease transmission to humans through consumption of raw milk. Moreover, the available diagnostic tools for detection C. burnetii infection are presented.

The main objective of this study was to assess the occurrence of Chlamydia spp. in Polish swine herds by using serological, molecular, and histological methods as well as by clinical manifestation of the suspected infection when reproductive disorders were taken into account. The seroprevalence among examined animals was 5.65% (96/1698), whereas molecular assessment of the pathogen was negative (0/298). The results of our investigation showed that Chlamydia spp. infection in swine may be suspected more often based on clinical manifestation or histological examination than serological or molecular methods.

The outbreak of chlamydiosis in one of the western provinces of Poland, was diagnosed accidentally as a concurrent infection in a commercial laying hen flock during an outbreak of fowl pox. For histological examination, skin and subcutaneous tissue samples from lesions on heads of the birds were collected. Swabs from throat and trachea have been examined by nested PCR, real-time PCR, and partial ompA sequencing. Detailed electron microscopy analysis revealed fowl pox intracytoplasmic inclusions, called Bollinger bodies, and the presence of other intracytoplasmic inclusions; specific for Chlamydia sp. Results of nested PCR confirmed the presence of Chlamydiaceae sp. in two tested samples. Surprisingly, one of the two Chlamydiaceae-positive cases turned out to be infected with a non-classified strain. Results of real-time PCR and sequencing confirmed the presence of a new Chlamydia species that has not been found in Poland to date. Partial sequencing and BLAST analysis of ompA gene sequence confirmed the highest homology to non-classified poultry strains of Chlamydia sp. that were previously detected in Germany and France. The zoonotic potential and the exact taxonomic status of this atypical strain have yet to be defined.

Diagnosis of Q fever in cattle is not easy due to the need to test the samples by both serological and molecular methods. Aim of this study was to evaluate qPCR, and phase I and II antibodies for detection of C. burnetii infection in cattle. A total of 187 bovine blood and vaginal swabs, and 97 milk samples, were tested. Limitations of serological tests were that the available indirect enzyme linked immunosorbent assay (iELISA) could lose positive results if antibody titres were low; or phase II antibodies were present. The highest level of correlation between iELISA and complement fixation test (CFT) was noted with the antigen specific phase I antibodies. Neither the mode of shedding nor its intensity correlated with phase I and II antibodies, but positive results in CFT mixed-phase and shedding in vaginal mucous did correlate, and showed the highest correlation. Antigenic diversity, and variability could be crucial in laboratory diagnosis of Q fever.

Mycoplasma bovis and Mycoplasma mycoides subspecies mycoides small colony (MmmSC) are causes of bovine mycoplasmosis and contagious bovine pleuropneumonia (CBPP), respectively, and are responsible for serious economic losses in cattle around the world. CBPP was last reported in Poland in 1939 but bovine mycoplasmosis is believed to be endemic. A survey of 3670 serum samples for antibodies to M bovis and MmmSC from 361 herds in 16 Polish provinces Poland between 2007 and 2010 found no evidence of CBPP. The seroprevalence of M bovis, however, appeared high with 76.7 per cent of samples giving a positive reaction in the ELISA test, which did not appear to reflect the clinical disease status of the cattle. Adjusting the sensitivity of the test reduced the prevalence to 28.2 per cent and reflects the levels reported in other European countries.

Bovine respiratory disease (BRD) complex is a major disease, classically in the indoor calves and feedlot young cattle. The etiopathogenesis of BRD is multifactorial and complex. In this complex etiology an equivalent role plays both the infectious agents as well as the environmental factors which are called also as environmental stressors (inappropriate livestock management like mistakes in animal nutrition, transport, handling, veterinary interventions etc.) (tab. 1). The most significant pathogens which are involved in the etiopathogenesis of BRD, i.e. suitable species of viruses such as (bovine respiratory syncytial virus (BRSV), parainfluenza virus type 3 (PI3V), bovine herpes virus type 1 (BHV1), bovine viral diarrhea virus (BVDV), are usually associated with concurrent bacterial infections represented by: Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and others (tab. 1) (Jared et al., 2010a, Kita et al., 1994, Klimentowski et al., 1995) and also mycoplasmal factors such as: Mycoplasma bovis, Mycoplasma bovirhinis, Mycoplasma dispar, Ureaplasma diversum and even Mycoplasma canis (Szymanska et al., 2010). Among bacteria M. haemolytica and P. multocida have been traditionally considered as the most common bacterial infectious factors in the BRD etiology (Fulton et al.; Reggiardo et al., 2005). On the other hand the one of the most often isolated mycoplasmal factors from BRD cases is Mycoplasma bovis. Its adaptative ability to a host organism increases owing to different versions of the same vsp gene family which encode particular adhesive factors of the mycoplasma, i.e. variable surface proteins (Vsps; Razin et al., 1998). The variability of vsp gene based on DNA transposition (Lysnyansky, 1996). Therefore the high Vsp variation determines the phenotypic variability of the antigen (Rosengarten et al., 1994) which further increases the virulence of the pathogen. M. bovis possess the ability to immunomodulate host defence against infection (Razin et al., 1998), for example inducing a synthesis of proinflammatory agents, i.e. TNF-α and nitric oxide (Jungi, et al., 1996) or stimulation of some acute phase protein production, such as haptoglobulin and serum amyloid A, which represent one of the most important components of acute phase response for cattle (Dudek, 2010). It is known that M. bovis acts as a stimulant on major subsets of T-lymphocytes (Vanden Bush, 2003). In contrast, some data show inhibitory properties of the pathogen which come down to its suppressive effect on lymphocytes (Vanden Bush & Rosenbusch, 2002; Thomas et al., 1990)

Pleomorphic bacteria are classified in three groups: large cell variants (LCV), small-cell variants (SVC) and small dense cells (SDC). The SCVs and the SDCs are the most frequent forms in the host and certainly (the most) resistant forms in the environment. The LCV form of Coxiella burnetii, which shares features common with gram-negative bacteria, diffuse chromatin and process clearly distinguishable outside and cytoplasmic membranes with LPS exposed on the surface. The LCVs are larger, more pleomorphic and metabolically active than the SCVs and the SDCs. The SDCs have been visualised in LCV as endospores, and they may be liberated upon the lysis of LCV or binary transverse fission with unequal cell division. The SDCs alone have never been isolated, free-living amoeba can provide an intra-cellular niche for SDC formation and survival of Coxiella burnetii in the environment. Binary transverse fission was observed both in the SCVs and the LCVs in cells cultured. The form changes within lifecycle of Coxiella burnetii are strategy to survive (Arricau-Bouvery & Rodolakis, 2005).

Q fever is an infectious disease caused by Coxiella burnetii. Diagnosis of Q fever based on clinical symptoms is unattainable; thus, different laboratory techniques are used to detect the infection. The aim of the study was to compare the diagnostic potential of ELISA, CFT, conventional PCR, real-time PCR and cell culture. The tests were carried out on 2251 serum samples from ruminants. Moreover, 668 placentas, 1277 vaginal swabs and 306 specimens of the internal organs of aborted foetuses were examined by PCR and cell culture. Pearson's chi-square test was used to compare the results obtained by ELISA, CFT, PCR, real-time PCR and isolation in cell culture. The χ(2) test confirmed that in most cases the results obtained by means of the different methods were correlated with each other (P