New insight on chlamydiae.

This article provides an overview of the current knowledge on chlamydiae, which are intracellular bacteria belonging to the Chlamydiaceae family. Whole-genome sequencing leads to great increases in the available data about Chlamydia spp. Recently, novel chlamydial taxons in various hosts living in different environments have been recognised. New species and taxons with Candidatus status have been recorded mainly in birds and reptiles. Chlamydia gallinacea is an emerging infectious agent in poultry with indirectly confirmed zoonotic potential. Recently, a new group of avian C. abortus strains with worldwide distribution in various wild bird families has been described. The definition of C. abortus species became outdated with the discovery of these strains and has been amended. It now includes two subgroups, mammalian and avian, the latter including all isolates hitherto referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates.

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.

Non-Invasive Sampling in the Aspect of African Swine Fever Detection-A Risk to Accurate Diagnosis.

African swine fever remains one of the most economically important and dangerous diseases of the Suidae family. Until now, neither a safe vaccine nor a treatment against ASF has been available, which is why prevention of the disease involves biosecurity measures and early recognition based on accurate diagnosis. Nowadays, different strategies for ASF detection are discussed to reduce both animal suffering and the costs of ASF surveillance. This article aims to indicate the risk, with regard to non-invasive sampling, for the detection of ASFV. In this study, we analyzed data from three independent animal trials, in the framework of the detection of positive samples in different matrices (blood, sera, oral and rectal swabs) collected from nineteen domestic pigs infected with similar doses but under different scenarios, including different ASFV strains or routes of infection. Genetic material of ASFV was found in all matrices, but detection occurred earlier in the blood samples than in the oral and the rectal swabs. Furthermore, analyses revealed that at relevant sampling timepoints, PCR-positive blood samples were detected more frequently and reached higher percentages (up to 100% during fever) than oral and rectal swabs. Moreover, mean Ct values in blood samples collected from animals infected with virulent strains were significantly lower than in oral and rectal swabs, ensuring a higher probability of ASFV detection. High Ct values and occasional shedding in all tested matrices, in the cases of animals infected by an attenuated ASFV-strain, showed that blood sampling may be necessary to confirm the presence of anti-ASFV antibodies in sera. This study showed that during veterinary surveillance, blood sampling (for both PCR and serological analyses) is essential for the accurate diagnosis of ASF and provides the highest probability of detection of the disease.

African Swine Fever (ASF) Trend Analysis in Wild Boar in Poland (2014-2020).

African swine fever (ASF) is a lethal hemorrhagic disease of Suidae, i.e., domestic pigs and wild boars. The disease was introduced to Poland in 2014 and is now present in the wild boar population. Appropriate ASF prevention requires further research for answers to fundamental questions about the importance of vectors in virus transmission, the impact of environmental factors on the presence of ASFV in wild boar habitats, and the role of survivors as potential virus carriers and their part in the potential endemicity of ASF. In order to analyze the changes in the molecular and serological prevalence of ASFV in wild boar population in Poland, real-time PCR and ELISA/IPT tests were conducted. In the analyzed period (2014-2020), most of the ASF-positive wild boars were molecular/virus-positive, however, over the years the percentage and the number of seropositive animals has increased. At the beginning of the epidemic, the disease was limited to a small area of the country. Since then, it has spread to new provinces of Poland. From the beginning and until today, most notifications of ASF-positive wild boars were for carcasses (passive surveillance), however, the number of serologically positive animals is still increasing. Despite the fact that notifications of ASF outbreaks are still being received near the eastern border of Poland, the old ASF area seems to be limited mainly to ASF serologically positive animals, which may indicate the beginning of ASF endemicity in Poland.

Highly Pathogenic Avian Influenza H5Nx in Poland in 2020/2021: a Descriptive Epidemiological Study of a Large-scale Epidemic.

Introduction: Highly pathogenic avian influenza (HPAI) outbreaks caused by the Gs/Gd lineage of H5Nx viruses occur in Poland with increased frequency. The article provides an update on the HPAI situation in the 2020/2021 season and studies the possible factors that caused the exceptionally fast spread of the virus. Material and Methods: Samples from poultry and wild birds delivered for HPAI diagnosis were tested by real-time RT-PCR and a representative number of detected viruses were submitted for partial or full-genome characterisation. Information yielded by veterinary inspection was used for descriptive analysis of the epidemiological situation. Results: The scale of the epidemic in the 2020/2021 season was unprecedented in terms of duration (November 2020-August 2021), number of outbreaks in poultry (n = 357), wild bird events (n = 92) and total number of affected domestic birds (approximately ~14 million). The major drivers of the virus spread were the harsh winter conditions in February 2020 followed by the introduction of the virus to high-density poultry areas in March 2021. All tested viruses belonged to H5 clade 2.3.4.4b with significant intra-clade diversity and in some cases clearly distinguished clusters. Conclusion: The HPAI epidemic in 2020/2021 in Poland struck with unprecedented force. The conventional control measures may have limited effectiveness to break the transmission chain in areas with high concentrations of poultry.

Screening for Coxiella Burnetii in Dairy Cattle Herds in Poland.

Introduction: The intracellular bacterium Coxiella burnetii is the aetiological agent of Q fever, a zoonosis affecting many animal species worldwide. Cattle and small ruminants are considered the major reservoirs of the bacteria and they shed it through multiple routes. Material and Methods: A total of 2,180 sera samples from 801 cattle herds in all Polish voivodeships were tested by ELISA for the presence of specific antibodies. Milk samples were obtained from seropositive cows in 133 herds as part of a separate study. The milk samples were examined by ELISA and real-time PCR tests. Results: Seroprevalence at the animal level was 7.06% and true positive seroprevalence was 6.0% (95% confidence interval (CI) 1.1-9.4). Seroprevalence at the herd level was estimated at 11.1% and true positive seroprevalence was 10.5% (95% CI 3.2-15.8). Shedding of the pathogen in milk was detected by real-time PCR in 33 out of 133 tested herds (24.81%, 95% CI 17.74-33.04%) and the presence of C. burnetii antibodies was confirmed in 85 of them (63.9%, 95% CI 55.13-72.05%). The highest level of conformity between ELISA and real-time PCR results was obtained for bulk tank milk samples. Conclusion: Coxiella burnetii infections are quite common in cattle herds across the country, which emphasises the crucial roles of surveillance and adequate biosecurity measures in the prevention and limitation of Q fever spread in Poland.

Dynamics of the African Swine Fever Spread in Poland.

Introduction: African swine fever (ASF) is a lethal haemorrhagic disease of Suidae, present in Poland since 2014. The natural reservoir of ASF in Europe is the wild boar (Sus scrofa); however, human activity facilitates long-distance introductions of the disease. In ASF control it is important to identify areas at increased risk of infection. Such identification and estimation of the disease's progress and subsequent spread will help to identify the specific preventive action needs in given zones. Serving this purpose, this study is a spatial and statistical analysis of ASF spread through noted outbreak data. Material and Methods: The spatial-temporal analysis was conducted on the basis of data including the time and location of all ASF outbreaks both in wild boars and domestic pigs in Poland in 2014-2021. Results: The analysis indicates possible routes and directions for further ASF spread in Poland, estimates the annual increase of the affected area (approx. 25,000 km2 every year since 2017) and marks trends. The strong method-independent correlation between the year and the surface area affected by African swine fever indicated a near-linear generalised trend. Conclusion: Given the growth trend, we can expect ASF to expand further into new territories of the country; however, it is important to realise that there is still a significant area to protect, because 60% of Poland remains ASF-free.

SARS-CoV-2 Monitoring on Mink Farms in Poland.

Introduction: Many countries have reported severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infections in mink, and transmission back to humans has raised the concern of novel variants emerging in these animals. The monitoring system on Polish mink farms detected SARS-CoV-2 infection first in January 2021 and has been kept in place since then. Material and Methods: Oral swab samples collected between February 2021 and March 2022 from 11,853 mink from 594 farms in different regions of Poland were screened molecularly for SARS-CoV-2. Isolates from those with the highest loads of viral genetic material from positive farms were sequenced and phylogenetically analysed. Serological studies were also carried out for one positive farm in order to follow the antibody response after infection. Results: SARS-CoV-2 RNA was detected in mink on 11 farms in 8 out of 16 Polish administrative regions. Whole genome sequences were obtained for 19 SARS-CoV-2 strains from 10 out of 11 positive farms. These genomes belonged to four different variants of concern (VOC) - VOC-Gamma (20B), VOC-Delta (21J), VOC-Alpha (20I) and VOC-Omicron (21L) - and seven different Pango lineages - B.1.1.464, B.1.1.7, AY.43, AY.122, AY.126, B.1.617.2 and BA.2. One of the nucleotide and amino acid mutations specific for persistent strains found in the analysed samples was the Y453F host adaptation mutation. Serological testing of blood samples revealed a high rate of seroprevalence on the single mink farm studied. Conclusion: Farmed mink are highly susceptible to infection with SARS-CoV-2 of different lineages, including Omicron BA.2 VOC. As these infections were asymptomatic, mink may become an unnoticeable virus reservoir generating new variants potentially threatening human health. Therefore, real-time monitoring of mink is extremely important in the context of the One Health approach.

Experimental inoculation of chicken broilers with C. gallinacea strain 15-56/1.

Chlamydia gallinacea is one of the new Chlamydia species, encountered predominantly in birds and occasionally in cattle, and its dissemination, pathogenicity and zoonotic potential have not yet been fully elucidated. Until now, no case of clinical infection has been described in poultry, but the number of studies is limited. This study was conducted to evaluate the course of infection and the impact on production parameters in chicken broilers inoculated with the strain 15-56/1 isolated from a Polish flock. The presence of C. gallinacea was confirmed in oropharyngeal and cloacal swabs by real-time PCR from the fifth day post inoculation (dpi). Pathogen DNA was also detected in many internal organs of inoculated chickens. All infected animals remained asymptomatic during the entire experimental period, although statistical analyses showed that broilers in the experimental group exhibited significantly lower body weight gains and feed conversion ratios than animals in the control group. These data indicate that subclinical C. gallinacea infection in broilers may lead to financial losses for poultry farmers.

Whole Genome Sequencing and Comparative Genome Analyses of Chlamydia abortus Strains of Avian Origin Suggests That Chlamydia abortus Species Should Be Expanded to Include Avian and Mammalian Subgroups.

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.

Synthesis of novel indole-thiazolidinone hybrid structures as promising scaffold with anticancer potential.

A series of novel indole-azolidinone hybrids has been synthesized via Knoevenagel reaction of 5-fluoro-3-formyl-1H-indole-2-carboxylic acid methyl ester and some azolidinones differing in heteroatoms in positions 1, 2 and 4. Their anticancer activity in vitro was screened towards MCF-7 (breast cancer), HCT116 (colon cancer), HepG2 (hepatoma), HeLa (cervical cancer), A549 (lung cancer), WM793 (melanoma) and THP-1 (leukemia) cell lines, and a highly active 5-fluoro-3-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-1H-indole-2-carboxylic acid methyl ester (3a) was identified and subjected to in-depth investigation of cytotoxicity mechanisms. This compound was found to possess the highest cytotoxic action towards tumor cells comparing with the action of other derivatives (1, 3b, 3c, 3d, 3e). Compound 3a exhibited toxicity toward MCF-7, HCT116, and A549, HepG2 cancer cells, while the non-malignant cells (human keratinocytes of HaCaT line and murine embryonic fibroblasts of Balb/c 3T3 line) possessed moderate sensitivity to it. The compound 3a induced apoptosis in studied tumor cells via caspase 3-, PARP1-, and Bax-dependent mechanisms; however, it did not affect the G1/S transition in HepG2 cells. The compound 3a impaired nuclear DNA in HepG2, HCT116, and MCF-7 cells without intercalating this biomolecule, but much less DNA damage events were induced by 3a in normal Balb/c 3T3 fibroblasts compared with HepG2 carcinoma cells. Thus, 5-fluoro-3-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-1H-indole-2-carboxylic acid methyl ester 3a was shown to trigger DNA damage and induce apoptosis of human tumor cells and it might be considered as an anticancer agent perspective for in-depth studies.

Molecular detection of Coxiella burnetii in small ruminants and genotyping of specimens collected from goats in Poland.

BACKGROUND: Coxiella burnetii is 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 of Coxiella burnetii in 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. RESULTS: Overall, 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 IS1111 element. C. burnetii infection 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. CONCLUSION: This study confirmed the relatively common occurrence of Coxiella burnetii in small ruminant herds in Poland. Interestingly, all genotyped samples represent cattle-associated MLVA genotypes.

African Swine Fever in Wild Boar (Poland 2020): Passive and Active Surveillance Analysis and Further Perspectives.

African swine fever (ASF) is a fatal hemorrhagic disease of wild boar and domestic pigs which has been present in Poland since 2014. By 2020, the ASF virus (ASFV) spread across Central, Eastern and Western Europe (including Germany), and Asian countries (including China, Vietnam, and South Korea). The national ASF eradication and prevention program includes continuous passive (wild boar found dead and road-killed wild boar) and active (hunted wild boar) surveillance. The main goal of this study was to analyze the dynamic of the spread of ASF in the wild boar population across the territory of Poland in 2020. In that year in Poland, in total 6191 ASF-positive wild boar were declared. Most of them were confirmed in a group of animals found dead. The conducted statistical analysis indicates that the highest chance of obtaining an ASF-positive result in wild boar was during the winter months, from January to March, and in December 2020. Despite the biosecurity measures implemented by holdings of domestic pigs, the disease also occurred in 109 pig farms. The role of ASF surveillance in the wild boar population is crucial to apply more effective and tailored measures of disease control and eradication. The most essential measures to maintain sustainable production of domestic pigs in Poland include effective management of the wild boar population, along with strict implementation of biosecurity measures by domestic pig producers.

Mink SARS-CoV-2 Infection in Poland - Short Communication.

INTRODUCTION: Since April 2020, when the first SARS-CoV-2 infection was reported in mink and subsequently in mink farm workers in the Netherlands, it has been confirmed that human-to-mink and mink-to-human transmission can occur. Later, SARS-CoV-2 infections in mink were reported in many European and North American countries. MATERIAL AND METHODS: Samples from 590 mink from a total of 28 farms were tested by real-time RT-PCR. Whole genome sequences from one positive farm were generated and genetic relatedness was established. RESULTS: SARS-CoV-2 RNA was detected on a breeder farm with stock of 5,850 mink. Active viraemia was confirmed in individually tested samples with Ct values respectively between 19.4 and 29.6 for E and N gene fragments. Further testing of samples from culled animals revealed 70% positivity in throat swabs and 30% seropositivity in blood samples. Phylogenetic analysis of full-length nucleotide sequences of two SARS-CoV-2 isolates revealed that they belong to the 20B Nextstrain clade. Several nucleotide mutations were found in analysed samples compared to the reference Wuhan HU-1 strain and some of them were nonsynonymous. CONCLUSION: We report the infection of mink with SARS-CoV-2 on one farm in Poland and the results of subsequent analysis of virus sequences from two isolates. These data can be useful for assessment of the epidemiological situation of SARS-CoV-2 in Poland and how it endangers public health.

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.

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.

Highly Pathogenic Avian Influenza H5N8 in Poland in 2019-2020.

INTRODUCTION: Repeated incursions of highly pathogenic avian influenza virus (HPAIV) H5 subtype of Gs/GD lineage pose a serious threat to poultry worldwide. We provide a detailed analysis of the spatio-temporal spread and genetic characteristics of HPAIV Gs/GD H5N8 from the 2019/20 epidemic in Poland. MATERIAL AND METHODS: Samples from poultry and free-living birds were tested by real-time RT-PCR. Whole genome sequences from 24 (out of 35) outbreaks were generated and genetic relatedness was established. The clinical status of birds and possible pathways of spread were analysed based on the information provided by veterinary inspections combined with the results of phylogenetic studies. RESULTS: Between 31 December 2019 and 31 March 2020, 35 outbreaks in commercial and backyard poultry holdings and 1 case in a wild bird were confirmed in nine provinces of Poland. Most of the outbreaks were detected in meat turkeys and ducks. All characterised viruses were closely related and belonged to a previously unrecognised genotype of HPAIV H5N8 clade 2.3.4.4b. Wild birds and human activity were identified as the major modes of HPAIV spread. CONCLUSION: The unprecedentedly late introduction of the HPAI virus urges for re-evaluation of current risk assessments. Continuous vigilance, strengthening biosecurity and intensifying surveillance in wild birds are needed to better manage the risk of HPAI occurrence in the future.

Chlamydiae - What's New?

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.

Animal Coronaviruses in the Light of COVID-19.

Coronaviruses are extremely susceptible to genetic changes due to the characteristic features of the genome structure, life cycle and environmental pressure. Their remarkable variability means that they can infect many different species of animals and cause different disease symptoms. Moreover, in some situations, coronaviruses might be transmitted across species. Although they are commonly found in farm, companion and wild animals, causing clinical and sometimes serious signs resulting in significant economic losses, not all of them have been classified by the World Organization for Animal Health (OIE) as hazardous and included on the list of notifiable diseases. Currently, only three diseases caused by coronaviruses are on the OIE list of notifiable terrestrial and aquatic animal diseases. However, none of these three entails any administrative measures. The emergence of the SARS-CoV-2 infections that have caused the COVID-19 pandemic in humans has proved that the occurrence and variability of coronaviruses is highly underestimated in the animal reservoir and reminded us of the critical importance of the One Health approach. Therefore, domestic and wild animals should be intensively monitored, both to broaden our knowledge of the viruses circulating among them and to understand the mechanisms of the emergence of viruses of relevance to animal and human health.

Attempts at the Development of a Recombinant African Swine Fever Virus Strain with Abrogated EP402R, 9GL, and A238L Gene Structure using the CRISPR/Cas9 System.

INTRODUCTION: African swine fever (ASF) is a pressing economic problem in a number of Eastern European countries. It has also depleted the Chinese sow population by 50%. Managing the disease relies on culling infected pigs or hunting wild boars as sanitary zone creation. The constraints on the development of an efficient vaccine are mainly the virus' mechanisms of host immune response evasion. The study aimed to adapt a field ASFV strain to established cell lines and to construct recombinant African swine fever virus (ASFV) strain. MATERIAL AND METHODS: The host immune response modulation genes A238L, EP402R, and 9GL were deleted using the clustered regularly interspaced short palindromic repeats/caspase 9 (CRISPR/Cas9) mutagenesis system. A representative virus isolate (Pol18/28298/Out111) from Poland was isolated in porcine primary pulmonary alveolar macrophage (PPAM) cells. Adaptation of the virus to a few established cell lines was attempted. The plasmids encoding CRISPR/Cas9 genes along with gRNA complementary to the target sequences were designed, synthesised, and transfected into ASFV-infected PPAM cells. RESULTS: The reconstituted virus showed similar kinetics of replication in comparison to the parent virus isolate. CONCLUSION: Taking into account the usefulness of the developed CRISPR/Cas9 system it has been shown that modification of the A238L, EP402R, and 9GL genes might occur with low frequency, resulting in difficulties in separation of various virus populations.

Sub-Saharan Africa and Eurasia Ancestry of Reassortant Highly Pathogenic Avian Influenza A(H5N8) Virus, Europe, December 2019.

We report detection of a highly pathogenic avian influenza A(H5N8) clade 2.3.4.4b virus in Europe. This virus was generated by reassortment between H5N8 subtype virus from sub-Saharan Africa and low pathogenicity avian influenza viruses from Eurasia.