Correction to: Detection of fowl adenovirus D strains in wild birds in Poland by Loop-Mediated Isothermal Amplification (LAMP).

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Detection of fowl adenovirus D strains in wild birds in Poland by Loop-Mediated Isothermal Amplification (LAMP).

BACKGROUND: The present study on the role of strains of adenovirus in wildlife reservoirs, and their prevalence is under exploration. In several previous studies, the presence of adenovirus strains in wild birds has been investigated. Worldwide distribution and outbreaks of adenovirus infections have been reported by many authors. The present study investigated the prevalence of FAdVs in 317 samples of different bird species from the northwestern region of Poland. An applied specific, sensitive, and efficient, without cross-reactivity loop-mediated isothermal amplification (LAMP) method to gauge the prevalence of fowl adenovirus strains in wild birds was developed and used. RESULTS: The method was based on the sequence of the loop L1 HVR1-4 region of the hexon gene of the FAdV genome reference strains FAdV-2 KT862805 (ANJ02325), FAdV-3 KT862807 (ANJ02399) and FAdV-11 KC750784 (AGK29904). The results obtained by LAMP were confirmed by real-time PCR. Among 317 samples obtained from wild birds, eight FAdV isolates (2.52%) were identified and produced a cytopathic effect (CPE) in chicken embryo kidney cells (CEK). Three FAdV types belonging to species Fowl adenovirus D were detected, which were isolated from three adenovirus types 2/3/11, and have been confirmed in three mute swans (Cygnus olor), three wild ducks (Anas platyrhynchos), one owl (Strigiformes), and one common wood pigeon (Columba palumbus). CONCLUSIONS: This study provides the first accurate quantitative data for the replication of fowl adenovirus strains in wild birds in Poland, indicating adenovirus interspecies transmission, and demonstrating the circulation of FAdVs in wild birds.

Hemorrhagic nephritis and enteritis in a goose flock in Poland--disease course analysis and characterization of etiologic agent.

Hemorrhagic nephritis enteritis of geese (HNEG) is an epizootic viral disease caused by infection with goose hemorrhagic polyomavirus (GHPV) that affects domestic geese. This study describes the epizootic analysis, laboratory diagnosis, and molecular characterization of GHPV isolates associated with HNEG cases in Poland. HNEG symptoms persisted in infected flocks for 2 wk with a 32% mortality rate. Primary gross lesions included hemorrhaging of the kidneys, intestines, and lungs. Histopathologic examination confirmed HNEG and identified that the causative agent was similar to other GHPV isolates and identical to the Toulouse 2008 isolate.

Probiotic supplementation as an alternative to antibiotics in broiler chickens.

Introduction: The broiler chicken digestive tract microbiome maintains the bird's immunity. Its composition has been shown to be important not only for the immune system but also for the gastrointestinal function and productivity of broiler chickens. If the microbiome is populated by supplementation with Lactobacillus, Pediococcus and Saccharomyces spp. - microorganisms with probiotic properties and alternatives to antibiotics - the immune system is stimulated. The use of probiotic supplements in the broiler production cycle can boost bird immunity and prevent adenovirus infection. The resilience of broiler chickens in different feeding schemes including supplementation with these microorganisms was assessed. Material and Methods: Four groups of Ross 308 chickens vaccinated on the standard scheme were investigated over 42 days. Group P received probiotics, prebiotics and vitamins; group AO received antibiotics; group P&AO received probiotics, prebiotics, vitamins and antibiotics; and the control group C received none of these. The birds' immunocompetence against common viral poultry pathogens and their immune response to an experimental challenge with a field strain of infectious bronchitis was evaluated by ELISA and production parameters were recorded. Results: Mortality was only observed in the control group and was 10%. All birds from the P, P&AO and AO groups responded to the challenge as would be expected of appropriately immunised chickens. Conclusion: The obtained results indicated that supplementation with synbiotic products and vitamins can enhance broiler chicken immunity and result in better production parameters.

Molecular characteristics of fowl adenovirus strains detected in broiler chickens on diets without immunostimulant supplements.

Introduction: Outbreaks of fowl adenovirus (FAdV) infection in chicken flocks in Poland threaten birds' health and lives and are rising in frequency. The risk of these infections in immunocompromised poultry flocks with developed clinical symptoms was analysed through virus detection in broiler chicks and correlation of cases with the birds' immune strength. Material and Methods: Samples were analysed from four broiler farms with chicks from the same hatchery in Silesia, Poland where feeding regimes were different. A normal diet was provided to birds on the control farm; a normal diet and probiotic, prebiotic, vitamin and microelement supplementation was supplied on another farm; a normal diet and antibiotics on the third; and a normal diet and both forms of supplementation were given on the fourth farm. Amplification of the virus DNA in a PCR with hexon gene L1 loop hypervariable region 1-4 primers determined the molecular characteristics of isolates of adenovirus strains obtained from necropsy tissue samples. The amplicon sequences were analysed, the pair-wise distances were determined, the maximum likelihood estimate for the gamma parameter for site rates was produced, Tajima's D neutrality test was run and the relative synonymous codon usage and transition/transversion bias were calculated. Results: Two species and two serotypes of fowl adenovirus - MW353018-FAdV-1/A-L-liver and MW353019-FAdV-5/B-I-intestine - were isolated in three-week-old broiler chicks on the control farm. Conclusion: Supplementation of broiler chicken flocks with probiotics, prebiotics, vitamins and microelements may have a significant beneficial effect on immunity and can prevent virus infection. The studies provided new information on the molecular characteristics of adenovirus strains isolated from chicks with a low level of immunity.

Comparison of loop-mediated isothermal amplification and PCR for the detection and differentiation of Marek's disease virus serotypes 1, 2, and 3.

The previously conducted study on loop-mediated isothermal amplification (LAMP) has shown its usefulness for the detection of Marek's disease virus (MDV) virulent field strains. The current study improves the previously designed LAMP method with an additional pair of loop primers, which accelerates the reaction, and describes two other LAMP procedures for the specific detection of FC126 strain of turkey herpesvirus and nonpathogenic SB-1 strain. The developed LAMP procedures were also confirmed and compared with PCR. Each LAMP reaction used three pairs of specific primers designed to target the nucleotide sequence of the very virulent MDV strain, the SB-1 strain of MDV-2, and turkey herpesvirus, respectively. All LAMP reactions were flexible and provided reliable results at a wide range of incubation temperatures from 54.0 to 62.3 C in 15 to 90 min. LAMP does not need any thermocyclers, because all assays were conducted in a water bath. The green fluorescence signal was recorded under ultraviolet illumination in LAMP samples containing virulent MDV and turkey herpesvirus where SYBR Green was added to the reaction mixture, whereas the SB-1-positive samples presented orange illumination after GelRed staining solution. The sensitivity of the three LAMP reactions ranged from 2 log10 plaque-forming units (PFU)/ml of the virulent MDV HPRS-16 strain and turkey herpesvirus (HVT) to 3 log10 PFU/ml of the SB-1 nonpathogenic strain. The sensitivity of the compared PCR was lower by 1-2 log10 PFU/ml. The conducted studies have shown that developed LAMP methods may be used instead of PCR for the detection and differentiation of virulent and nonpathogenic MDV strains used in prophylaxis against MD. LAMP may be conducted without access to thermocyclers.

Molecular analysis Polish isolates of goose hemorrhagic polyomavirus from geese and free-living birds.

Goose haemorrhagic polyomavirus (GHPV) is the viral agent of hemorrhagic nephritis and enteritis of geese (HNEG), a lethal disease of goose. The study describes the results of a molecular analysis Polish isolates of GHPV from geese and free-living birds based on complete VP1 gene and VP2 gene sequences. The sequences were analyzed and aligned with different GHPV isolates sequences accessible in the GenBank database. This study indicates affiliation GHPV isolates from fee-living birds and GHPV isolates circulating in Polish goose flocks and around the world to the same genetic groups, which proves their evolutionary relationship and indicates the potential role of free-living birds as a source of infections for poultry.

West Nile Virus Lineage 2 in Free-Living Corvus cornix Birds in Poland.

The summer temperatures recorded in Poland in 2022 were among the highest in over 30 years and, combined with higher-than-expected rainfall, gave the impression of an almost tropical climate. Such climatic conditions were ideal for the transmission of vector-borne zoonotic diseases such as West Nile fever. In northeastern Poland, in the Mazowieckie region, the Polish event-based surveillance network reported increased fatalities of free-living hooded crows (Corvus corone cornix). West Nile virus (WNV) lineage 2 was identified for the first time as the etiological agent responsible for the death of the birds. WNV was detected in 17 out of the 99 (17.17%) free-living birds tested in this study. All the WNV-infected dead birds were collected in the same area and were diagnosed in September by the NVRI and confirmed by the EURL for equine diseases, ANSES, in October 2022. Unnaturally high temperatures recorded in Poland in 2022 likely favored the infection and spread of the virus in the avian population. A nationwide alert and awareness raising of blood transfusion centers and hospitals was carried out to prevent human infections by WNV.

Loop-mediated isothermal amplification for the detection of goose circovirus.

BACKGROUND: Goose circovirus (GCV) presents an immunosuppressive problem in production of geese. The infection's clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR) and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP) as a simple method of GCV detection. RESULTS: The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRed(TM) solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods. CONCLUSIONS: The developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV.

Quantitative analysis of waterfowl parvoviruses in geese and Muscovy ducks by real-time polymerase chain reaction: correlation between age, clinical symptoms and DNA copy number of waterfowl parvoviruses.

BACKGROUND: Waterfowl parvoviruses cause serious loss in geese and ducks production. Goose parvovirus (GPV) is infectious for geese and ducks while Muscovy duck parvovirus (MDPV) infects Muscovy ducks only. So far, for these viruses' sensitive detection polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were applied. However, there was no molecular biology method for both waterfowl parvoviruses detection and quantification which could unify the laboratory procedures. The level of GPV and MDPV replication and distribution plays a significant role in the parvoviral infection progress and is strictly correlated to clinical symptoms. Meanwhile, experiments conducted previously on GPV distribution in geese, performed as animal trial, did not involve epidemiological data from the disease field cases. The study on the correlation between age, clinical symptoms and viral DNA copy number may be benefitable in understanding the GPV and MDPV infection. Such data may also aid in determination of the stage and severity of the infection with parvoviruses. Therefore the aim of this study was to develop quantitative real-time PCR for parallel detection of GPV and MDPV in geese and Muscovy ducks and to determine the correlation between the age of the infected birds, clinical symptoms and DNA copy number for the estimation of the disease stage or severity. RESULTS: In order to develop quantitative real-time PCR the viral material was collected from 13 farms of geese and 3 farms of Muscovy ducks. The designed primers and Taqman probe for real-time PCR were complementary to GPV and MDPV inverted terminal repeats region. The pITR plasmid was constructed, purified and used to prepare dilutions for standard curve preparation and DNA quantification. The applied method detected both GPV and MDPV in all the examined samples extracted from the heart and liver of the infected birds. The conducted correlation tests have shown relationship between age, clinical symptoms during parvoviral infection and the DNA copy number of these pathogens. The method allowed for a sensitive detection of GPV and MDPV even in 1-week old infected goslings or 2-week old ducklings before observation of any disease symptoms. CONCLUSIONS: The developed method was found to be a valuable tool for the unification of laboratory procedures and both parvoviruses parallel detection and quantification. The conducted analysis revealed significant correlation between the age of the infected birds, the observed clinical symptoms and DNA copy number of GPV and MDPV in the examined organs. The obtained data may aid in better understanding of the pathogenesis and epidemiology of Derzsy's disease and 3-w disease as well as estimation of the infection's severity and stage of the disease.

Current epidemiological situation in the context of inclusion body hepatitis in poultry flocks in Poland.

The research has been undertaken to understand the spreading of adenovirus strains in Poland's poultry flocks in the last six years. One hundred and forty-nine herds suspected of infection with adenoviruses were tested and the presence of poultry adenoviruses was found in 86 studied herds which were about 57,71% of examined flocks. Thirty-eight (44.18%) strains were connected with the infection of inclusion body hepatitis, 11 (12.79%) strains were isolated from digestive system dysfunction, 33 (38.37%) strains had been obtained from the flocks with no symptomatic changes/behaviour, and four (4.65%) strains were obtained from flocks with the manifestation of depression. Sequencing analysis was based on Loop L1 region of the HVR1-4 fragment of the hexon gene. The adenovirus strains were classified into five species FAdV-A-E, belonging to the following eight serotypes: FAdV-1/A, FAdV-5/B, FAdV-3/D, FAdV-2/11/D, FAdV-10/C, and FAdV-7/8a/E. The most common serotype in poultry turned out to be type/species FAdV-2/11/D, FAdV-5/B, and FAdV-7/8b/E while the least frequent was type/species FAdV-10/C (only two strains respectively of this type were isolated with the following range: FAdV-1/A 6 (6.97%), FAdV-5/B 24 (27,90%), FAdV-3/D 4 (4,65%), FAdV-10/C 2 (2,32%), FAdV-2/11/D 36 (41,86%), and FAdV-E 14 (16.27%). The understanding of genetic diversity, geographic distribution, and antigenic properties of fowl adenovirus strains (FAdVs) isolated in Poland have been evaluated.

A Preliminary Study of the Poultry Body Weight Effect of Carvacrol in Litter and Of Carvacrol Residue in Organ Tissue of Exposed Chickens.

Introduction: Carvacrol is an essential oil extracted from oregano which can be used as a natural additive in poultry litter and could have a positive impact not only on production rates but also on the quality of poultry meat. The aim of this study was to evaluate the effect of the addition of carvacrol to litter on weight gain and the occurrence of residues in chicken tissues. Material and Methods: One-day-old Ross 308 chicks were used for the study and were randomly divided into two experimental groups. For 42 days, one group was kept in a room with litter enriched with carvacrol and the second group was kept in a room with litter without carvacrol. After 42 days, the birds were sacrificed and necropsied. Carvacrol content was determined in homogenised organ tissue samples by liquid chromatography-mass spectrometry. Results: Weekly weighing results showed that exposure to carvacrol in litter had no impact on chicken body weight. The analysis of plasma, muscle, liver and lung tissue after 42 days' exposure clearly indicated that there were residues of carvacrol in the analysed matrices. Conclusion: Exposure of chickens to carvacrol left residues but did not affect body weight.

Rapid detection of Marek's disease virus in feather follicles by loop-mediated amplification.

Marek's disease (MD) remains a serious problem in the production of poultry. The disease is caused by Marek's disease virus (MDV), and despite the ubiquitous use of vaccination to control losses, MD still affects poultry farming worldwide. The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) method for the simple and inexpensive detection of MDV in feather tips of chickens. Two pairs of specific primers complementary to the meq oncogene of MDV were designed, targeting the sequence of the very virulent MDV strain, RB1B. Bst polymerase was used for the isothermal amplification of viral DNA at 65 C for 90 min in a water bath. The fluorescence signal was identified in MDV-positive samples after the addition of SYBR Green and ultraviolet (UV) illumination. The sensitivity of LAMP was 2 log 10 plaque-forming units (PFU)/ml of HPRS16 and 10(3) copies/il of plasmid containing the target gene (meq) and was equal in sensitivity to PCR amplification. Due to the use of three sets of primers, LAMP was highly specific for MDV-1 DNA. The developed LAMP technique is a rapid and simple tool for the specific detection of MDV in samples of feathers taken from live chickens. Since the use of thermocyclers is not necessary for LAMP assay, it can be conducted by small laboratories and even field veterinarians.

Attempts to detect West Nile virus in wild birds in Poland.

The aim of the study was to attempt the detection of West Nile virus (WNV) in wild birds in Poland. Forty-eight species of 1912 wild birds were used for the investigations. The birds were derived from various locations in Poland from early spring till late autumn of the years 2009-2011. The brain samples were homogenised and cellular RNA was isolated. Two methods (RT-PCR and nested RT-PCR) were used. The presence of WNV RNA was not detected in the samples examined. Additionally, a short analysis of the epizootiological situation regarding the presence of WNV in Poland is presented.

Fowl adenovirus strains 1/A and 11/D isolated from birds with reovirus infection.

Inclusion body hepatitis (IBH) is, in some cases, a fatal disease affecting fowl by adenovirus strains which are subdivided into 5 species (A-E). In the current study, we investigated sequences from the Loop L1 region of the hexon gene of sequences of adenovirus field stains 1/A and 11/D isolated from a poultry flock co-infected with IBH and avian reoviruses ARVs. In early 2021, an epidemiologic survey highlighted the coinfection adenoviruses with other viruses (orthoreovirus infection) as being particularly deleterious within the poultry industry. Here, we investigated the Loop L1 HVR1-4 region of the hexon gene with relative synonymous codon usage (RSCU) designation and RSCU inclusive of all the mutations. These are the first results that have been presented on fowl adenovirus species A and D with simultaneous reovirus infection in 38-days old broiler chickens in Poland.

Characterisation of adenovirus strains represented species B and E isolated from broiler chicken flocks in eastern Poland.

Fowl adenovirus strains were isolated from the internal organs of 3-wk-old broiler flocks exhibited clinical signs associated with inclusion body hepatitis (IBH). The isolated strains were molecularly characterised and sequencing revealed three distinct clusters. One cluster showed close proximity at the nucleotide level with adenovirus type/species - 6/E, 7/E, 8a/E, and 8b/E. The second cluster contained five reference sequences belonging to the species FAdV-D and E. A third cluster contained one field and four reference sequences belonging to the FAdV-5/B, FAdV-4/C, FAdV-2/D, and FAdV-1/A type/species respectively. The heterogenicity, Relative Synonymous Codon Usage (RSCU), codon composition, and nucleotide frequencies were examined. Statistical analyses, were carried out. The maximum likelihoods for the examined sequences were estimated. The data indicated that correlation between isolated of adenovirus type/species 5/B, and E in Poland have been presented. Indicated adenovirus types and their combinations with locally circulating FAdVs strains could have implications for current detection methods and pathogenicity on infected chickens.

Antimicrobial Resistance Glides in the Sky-Free-Living Birds as a Reservoir of Resistant Escherichia coli With Zoonotic Potential.

Antimicrobial resistance (AMR) is one of the most important global health concerns; therefore, the identification of AMR reservoirs and vectors is essential. Attention should be paid to the recognition of potential hazards associated with wildlife as this field still seems to be incompletely explored. In this context, the role of free-living birds as AMR carriers is noteworthy. Therefore, we applied methods used in AMR monitoring, supplemented by colistin resistance screening, to investigate the AMR status of Escherichia coli from free-living birds coming from natural habitats and rescue centers. Whole-genome sequencing (WGS) of strains enabled to determine resistance mechanisms and investigate their epidemiological relationships and virulence potential. As far as we know, this study is one of the few that applied WGS of that number (n = 71) of strains coming from a wild avian reservoir. The primary concerns arising from our study relate to resistance and its determinants toward antimicrobial classes of the highest priority for the treatment of critical infections in people, e.g., cephalosporins, quinolones, polymyxins, and aminoglycosides, as well as fosfomycin. Among the numerous determinants, bla CTX-M-15, bla CMY-2, bla SHV-12, bla TEM-1B, qnrS1, qnrB19, mcr-1, fosA7, aac(3)-IIa, ant(3")-Ia, and aph(6)-Id and chromosomal gyrA, parC, and parE mutations were identified. Fifty-two sequence types (STs) noted among 71 E. coli included the global lineages ST131, ST10, and ST224 as well as the three novel STs 11104, 11105, and 11194. Numerous virulence factors were noted with the prevailing terC, gad, ompT, iss, traT, lpfA, and sitA. Single E. coli was Shiga toxin-producing. Our study shows that the clonal spread of E. coli lineages of public and animal health relevance is a serious avian-associated hazard.

Occurrence of Marek's Disease in Poland on the Basis of Diagnostic Examination in 2015-2018.

INTRODUCTION: Marek's disease (MD) is a tumourous disease caused by Marek's disease virus (MDV) and most commonly described in poultry. The aim of the study was to determine the occurrence of Marek's disease virus infections in Poland and analyse clinical cases in the years 2015-2018. MATERIAL AND METHODS: The birds for diagnostic examination originated from 71 poultry flocks of various types of production. Birds were subjected to anatomopathological examination post mortem, during which liver and spleen sections and other pathologically changed internal organs were taken. These sections were homogenised with generally accepted methods, then total DNA was isolated and amplified with a real-time PCR. A pair of primers complementary to the MDV genome region encoding the meq gene were used. RESULTS: MDV infection was found predominantly in broiler chicken flocks (69.01%), and also in layer breeder (9.85%) and commercial layer flocks (7.04% each). CONCLUSION: The results of research conducted in the years 2015-2018 clearly indicate that the problem of MDV infections is still current.

Isolation and molecular characterization of Fowl adenovirus strains in Black grouse: First reported case in Poland.

This article describes the isolation, molecular characterization, and genotyping of two fowl adenovirus (FAdVs) strains with GenBank Accession numbers (MT478054, JSN-G033-18-L and MT478055, JSN-G033-18-B) obtained from the internal organs of black grouse (Lyrurus tetrix). This study also reveals the first confirmation of fowl adenovirus in Poland, supporting one of the hypotheses about the probability of fowl adenovirus interspecies transmission. The adenovirus strain sequences were investigated via phylogenetic analysis and were found to have an overall mean pairwise distance of 2.189. The heterogeneity, Relative Synonymous Codon Usage (RSCU), codon composition, and nucleotide frequencies were examined. Statistical analyses and Tajima's test for the examined sequences were carried out. The Maximum Likelihood for the examined sequences substitutions was performed. The results of the sequence analysis identified MT478054, JSN-G033-18-L and MT478055, JSN-G033-18-B as strains of fowl adenovirus 2/11/D, with the Fowl adenovirus D complete sequence showing a 93% match. Wild birds may act as a natural reservoir for FAdVs and likely play an important role in the spreading of these viruses in the environment. The findings reported here suggest horizontal transmission within and between avian species.

Elaboration of Triplex PCR for Detection of Selected Viral Infections in Waterfowl.

INTRODUCTION: Viral infections are the greatest threat to waterfowl and cause significant economic losses. Diagnosis and differentiation of three goose viruses is difficult in the field and often requires laboratory confirmation. Therefore, the aim of the study was to develop a triplex PCR and optimise its parameters for simultaneous detection of DNA of goose parvovirus (GPV), goose polyomavirus (GHPV), and goose circovirus (GoCV). MATERIAL AND METHODS: The DNA of viruses isolated from field cases from the National Veterinary Research Institute's own collection was used for the study. The primer attachment temperature, the number of reaction cycles, and the Taq DNA polymerase and Mg2+ concentrations were optimised. The sensitivity and specificity of this triplex PCR was also determined. RESULTS: Based on the obtained results, triplex PCR parameters were optimised for simultaneous detection of DNA of GPV, GHPV, and GoCV in one sample. The following PCR products of the expected size were obtained: GPV DNA of 806 bp, GoCV DNA of 571 bp, and GHPV DNA of 180 bp. CONCLUSION: The developed triplex PCR method proved to be useful for simultaneous detection of infections with three waterfowl viruses and will be used in relevant laboratory diagnostics.