Antibiotic resistance of Gallibacterium anatis biovar haemolytica isolates from chickens.

Introduction: Gallibacterium anatis is an opportunistic bacteria inducing a range of clinical signs in poultry. Gallibacterium anatis strains show multidrug resistance to antibacterial substances. The purpose of this study was to examine the susceptibility of G. anatis biovar haemolytica isolates collected from the respiratory, reproduction and gastrointestinal tracts of chickens to different antibiotics from various classes. Material and Methods: Gallibacterium anatis biovar haemolytica was identified in tracheal swab and gastrointestinal and reproductive tract tissue samples from Polish layer and broiler chicken flocks. Twenty six isolates with ß-haemolysis capability, each from a different flock, obtained from the respiratory (n = 8), reproductive (n = 10) and gastrointestinal (n = 8) tracts were selected and identified by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry after culturing. A PCR method targeting the 16S genes was used for verification of isolates. The isolates' susceptibility to 20 antimicrobials was evaluated using the disc diffusion method for 8 drugs and the dilution method for the other 12. In addition, they were tested for the presence of the GtxA, gyrB and flfA virulence genes and blaROB, aphA, tetB and tetH antibiotic resistance genes by PCR. Results: The most prevalent antibiotic resistance was to tilmicosin, tylosin and quinupristin/dalfopristin (all 100%), erythromycin (96.2%), tetracycline (96.2%), linezolid (92.3%) and teicoplanin (92.3%). Universal susceptibility was to only one antibiotic, chloramphenicol. Statistically significant differences were found between the resistance of gastrointestinal tract strains and that of strains from other tracts to daptomycin, gentamicin, ciprofloxacin and colistin. The GtxA and gyrB genes were detected in 100% of isolates and flfA in 19.2%. The isolates most frequently contained tetB and less frequently tetH and aphA, and did not contain blaROB. Conclusion: Most G. anatis biovar haemolytica isolates were resistant to many classes of antibiotics. Therefore, it is necessary and important to be vigilant for the occurrence of these bacteria and thorough in their diagnosis.

Case reports involving coinfection with Avibacterium paragallinarum and Ornithobacterium rhinotracheale in broiler chickens and Avibacterium endocarditis in broiler breeding hens in Poland.

ABSTRACTThe study describes three clinical cases of infection with Avibacterium spp.. In case no. 1, respiratory clinical signs and high mortality (0.7-4.2% daily; total 21.2%) in Ross 308 broiler chickens were shown to be caused by coinfection with sequence type 9 of O. rhinotracheale presumptive serotype A and A. paragallinarum presumptive serotype B. The identical (pulsed-field gel electrophoresis) restriction pattern (pulsotype) of seven A. paragallinarum isolates indicated that infectious coryza in broilers was caused by the same clone. In cases 2 and 3, sudden increased deaths in Ross 308 broiler breeders (especially males) with lesions in the endocardium (valvular or mural endocarditis) were shown to be caused by A. endocarditis. Among nine antibiotics tested, florfenicol was the only antibiotic to which all A. paragallinarum and O. rhinotracheale isolates were susceptible. Out of the eight antibiotics tested, 11 A. endocarditis isolates from both clinical cases of infective endocarditis were susceptible to penicillin, amoxicillin, doxycycline and florfenicol. The A. endocarditis isolates tested in both clinical cases had different PFGE patterns (pulsotypes), but identical within a case. The causes of infectious coryza and infective endocarditis in the cases presented have not been determined. In the prevention of infectious diseases in large-scale livestock farming, it is very important to follow the rules of biosecurity.

Mycoplasma gallisepticum and Mycoplasma synoviae in Turkeys in Poland.

The pathogenic mycoplasmas are among the bacteria causing significant losses in the poultry industry worldwide. Mycoplasma gallisepticum (MG) and M. synoviae (MS) are economically important pathogens causing chronic respiratory disease, decreased growth, egg production and hatchability rates, and significant downgrading of carcasses. Effective diagnosis of infection with these species in poultry is highly requisite considering their two routes of spreading-horizontal and vertical. Their prevalence and molecular epidemiology were investigated in 184 turkey flocks in Poland. Tracheal samples were selected from 144 broiler flocks and 40 turkey breeder flocks collected in 2015-2023. The prevalence of MG was determined by real-time PCR targeting the 16S rRNA gene and PCR targeting the mgc2 gene, and MS was determined by a 16-23S rRNA real-time PCR and a vlhA gene PCR. Further identification and molecular characterization were carried out using PCR and sequencing. M. gallisepticum and M. synoviae were found in 8.33% and 9.72% of turkey broiler flocks respectively. The phylogenetic analysis of MG isolates in most cases showed high similarity to the ts-11-like strains. MS isolates showed high similarity to strains isolated from flocks of laying hens causing EAA. Additional tests detected Ornithobacterium rhinotracheale, Gallibacterium anatis, Enterococcus faecalis and Enterococcus faecium, Staphylococcus aureus and Riemerella anatipestifer. These secondary pathogens could have significantly heightened the pathogenicity of the mycoplasma infections studied.

Prevalence, Identification and Antibiotic Resistance of Gallibacterium anatis Isolates from Chickens in Poland.

The Gram-negative bacterium Gallibacterium anatis is part of the normal avian respiratory, intestinal and reproductive tract microflora and can be transmitted horizontally and vertically. With the coexistence of other relevant factors, G. anatis becomes an opportunistic pathogen, economically damaging to the poultry industry. This bacterium's prevalence and molecular epidemiology were investigated, and the antimicrobial treatment options for G. anatis infection in chicken flocks in Poland were assessed. Tracheal samples from 182 flocks were collected between April 2022 and March 2023. The bacterial prevalence was determined by PCR targeting the gyrB gene and 16-23S rRNA. Gallibacterium anatis was identified by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF) after culturing and PCR amplification. Isolates' susceptibility to 11 antimicrobials was assessed with a disc diffusion test. Isolates were also tested for gyrB, GtxA and flfA virulence genes and blaROB, aphA, tetB and tetH antibiotic resistance genes by PCR. Forty-one flocks (22.5%) were positive through PCR. Antibiotic resistance was most frequently observed against tilmicosin, tylosin, enrofloxacin, amoxicillin, tetracycline and doxycycline. Multiple resistance to at least eight antibiotics occurred in 20% of isolates and to at least four in 100%. The occurrence of gyrB was noted in 100%, GtxA was detected in 89%, and flfA was found in 14% of positive samples. The tetB gene was present in 61.0% of positive samples, tetH was in 36.0%, aphA was in 16.7%, and blaROB was in 5.6%. Significant differences were found in G. anatis isolates related to the presence of the virulence genes GtxA and gyrB and the presence of resistance genes (p < 0.05) associated with resistance to tetracyclines, ß-lactams and aminoglycosides. The continued rise in the resistance of G. anatis to a broadening range of antibiotics is a major problem for the poultry industry worldwide, as well as for public health. The findings of this study may expand the knowledge of the pathogenicity of G. anatis in poultry.

Molecular Detection and Phylogenetic Analysis of Riemerella anatipestifer in Poultry and Wild Geese in Poland.

Riemerella anatipestifer (RA) is one of the most relevant bacterial pathogens of commercial waterfowl from clinical and economic points of view. Our study aimed to evaluate the prevalence of RA infection in different types of commercial poultry in Poland and verify the potential role of wild geese as vectors of this pathogen. We tested a total of 126 poultry flocks, including geese (N = 20), ducks (N = 42), turkeys (N = 64) and 19 wild geese, including greater white-fronted geese (N = 9), greylag geese (N = 5) and Taiga bean geese (N = 5). Tracheal swabs were examined for RA using a PCR targeting a conserved region of the 16S rRNA gene. Selected PCR products were sequenced to perform the phylogenetic analysis. Among the commercial poultry, the highest RA prevalence was found in flocks of ducks (35.7%) and geese (30.0%), whereas the lowest one was found in turkeys (3.2%). Most tested wild geese (94.7%) were RA positive. The phylogenetic analysis showed relatively low genetic diversity of the sequences analyzed, which gathered in two clusters of the phylogenetic tree, and the minimum nucleotide identity was 98.6%. Our results would support the contention that RA isolates from commercial poultry circulate in wild bird populations but are not transmitted back to poultry.

Multispectral Portable Fibre-Optic Reflectometer for the Classification of the Origin of Chicken Eggshells in the Case of Mycoplasma synoviae Infections.

The proper classification of the origins of food products is a crucial issue all over the world nowadays. In this paper, the authors present a device-a multispectral portable fibre-optic reflectometer and signal processing patch-together with a machine-learning algorithm for the classification of the origins of chicken eggshells in the case of Mycoplasma synoviae infection. The sensor device was developed based on previous studies with a continuous spectrum in transmittance and selected spectral lines in reflectance. In the described case, the sensor is based on the integration of reflected spectral data from short spectral bands from the VIS and NIR region, which are produced by single-colour LEDs and introduced to the sample via a fibre bundle. The measurement is carried out in a sequence, and the reflected signal is pre-processed to be put in the machine learning algorithm. The support vector machine algorithm is used together with three different types of data normalization. The obtained results of the F-score factor for classification of the origins of samples show that the percentages of eggs coming from Mycoplasma synoviae infected hens are up to 87% for white and 96% for brown eggshells.

Bacterial communities of the oviduct of turkeys.

Bacterial communities in the reproductive tract of avian species play an important role in keeping birds healthy and encouraging growth. Infection can occur during egg formation with pathogens that can be transmitted to the embryo. In this study, we investigated the bacterial composition in the turkey reproductive tract using a taxa identification based on the amplicon sequence of the V3-V4 region of the 16S rRNA gene. The microbial composition and relative abundance of bacteria differed between individual birds. Among the 19 phyla detected in turkey oviduct were unique taxa like Planctomycetes or Petescibacteria. Differences in composition of bacterial diversity were found at the family and genus level. Oviducts contained also several genus with well-recognized avian pathogens like Escherichia-Shigella, Enterococcus, Staphylococcus, and Ornithobacterium. Some of the bacteria described in this study have not been so far identified in turkeys. The objective of this study was to identify bacterial communities in the turkey oviduct and compared the composition of the oviduct with that in chickens broadening the knowledge of the microbial composition in the reproductive tract of poultry.

The Microbial Community of the Respiratory Tract of Commercial Chickens and Turkeys.

Respiratory tract health critically affects the performance of commercial poultry. This report presents data on the microbial community in these organs from a comprehensive study of laying chickens and turkey breeders. The main objective was to characterize and compare the compositions of the respiratory system bacteria isolated from birds of different ages and geographical locations in Poland. Using samples from 28 turkey and 26 chicken flocks, the microbial community was determined by 16S ribosomal RNA sequencing. There was great variability between flocks. The diversity and abundance of upper respiratory tract (URT) bacteria was greater in chickens than in turkeys. At the phyla level, the URT of the chickens was heavily colonized by Proteobacteria, which represented 66.4% of the total microbiota, while in turkeys, this phylum constituted 42.6% of all bacteria. Firmicutes bacteria were more abundant in turkeys (43.2%) than in chickens (24.1%). The comparison of the respiratory tracts at the family and genus levels showed the diversity and abundance of amplicon sequence variants (ASV) differing markedly between the species. Potentially pathogenic bacteria ASV were identified in the respiratory tract, which are not always associated with clinical signs, but may affect bird productivity and performance. The data obtained, including characterization of the bacterial composition found in the respiratory system, may be useful for developing effective interventions strategies to improve production performance and prevent and control disease in commercial laying chickens and turkeys.

Occurrence of Ornithobacterium Rhinotracheale in Polish Turkey Flocks.

Introduction: Ornithobacterium rhinotracheale (ORT) causes significant economic losses to the poultry industry around the world. The bacterium often affects poultry as part of multiple infections causing very serious clinical signs that are usually not limited only to the respiratory system. This study's main objective was the retrospective detection and identification of ORT in turkey flocks. Material and Methods: ORT identification was performed in 6,225 samples taken from 133 different flocks between 2015 and 2020. Molecular methods were used, specifically real-time PCR and traditional PCR. We focused on partial 16S rRNA gene sequences of isolates, which were compared with sequences obtained from GenBank. The reaction products were analysed phylogenetically. Molecular methods indicating secondary infections was carried out, and the bacterial composition of the upper respiratory tract was 16S metasequenced for selected flocks to identify any other pathogens. Results: The presence of ORT was detected in 30.83% of samples by real-time PCR and 28.57% by PCR. Phylogenetic analysis of the PCR products from the turkeys samples showed that their sequences resolved into two main genetic groups. Tests for the occurrence of secondary infections showed the presence of Mycoplasma gallisepticum and M. synoviae in some samples but the total absence of Bordetella avium. The upper respiratory tract in turkeys was dominated by two major phyla Firmicutes and Proteobacteria. At the genus level, the genera Ornithobacterium, Mycoplasma, Gallibacterium, Avibacterium, and Escherichia-Shigella were found which may include pathogenic bacteria that can cause clinical symptoms. Conclusion: The results of the analysis of multiple infection carried out in flocks with respiratory signs are probably associated with outbreaks of ornithobacteriosis in turkey flocks in Poland.

Evidence of Mycoplasma spp. transmission by migratory wild geese.

Mycoplasma infections have been found in different species of waterfowl worldwide. However, the question of how the pathogens have been transmitted and dispersed is still poorly understood. Samples collected from clinically healthy greater white-fronted geese (Anser albifrons) (N = 12), graylag geese (Anser anser) (N = 6), taiga bean geese (Anser fabalis) (N = 10), and barnacle geese (Branta leucopsis) (N = 1) were tested for Mycoplasma spp. All Mycoplasma-positive samples were specified by species-specific PCR for Mycoplasma anserisalpingitidis (formerly known as Mycoplasma sp. 1220), M. anseris, M. anatis, and M. cloacale. The presence of Mycoplasma spp. was confirmed in 22 of 29 sampled birds (75.9%). Mycoplasma anserisalpingitidis was the most frequently detected species (15 of 22, 68.2%). However, we did not detect any of the other Mycoplasma spp. typical for geese, among which are M. anatis, M. anseris, and M. cloacale. Phylogenetic analysis revealed that Polish sequences of M. anserisalpingitidis formed a distinct branch, along with 2 Hungarian isolates obtained from domestic geese. Eight of the samples identified as Mycoplasma spp.-positive were negative for the aforementioned Mycoplasma species. A phylogenetic tree constructed based on partial 16S rRNA gene analysis showed that Mycoplasma spp. sequences collected from Polish wild geese represent a distinct phylogenetic group with Mycoplasma sp. strain 2445 isolated from a domestic goose from Austria. The results of our study showed that wild geese could be a reservoir and vector of different species of the Mycoplasma genus that can cause significant economic losses in the domestic goose industry.

Occurrence of Mycoplasma spp. in wild birds: phylogenetic analysis and potential factors affecting distribution.

Different Mycoplasma species have been reported in avian hosts. However, the majority of studies focus on one particular species of Mycoplasma or one host. In our research, we screened a total of 1141 wild birds representing 55 species, 26 families, and 15 orders for the presence of mycoplasmas by conventional PCR based on the 16S rRNA gene. Selected PCR products were sequenced to perform the phylogenetic analysis. All mycoplasma-positive samples were tested for M. gallisepticum and M. synoviae, which are considered the major pathogens of commercial poultry. We also verified the influence of ecological characteristics of the tested bird species including feeding habits, habitat types, and movement patterns. The presence of Mycoplasma spp. was confirmed in 498 birds of 29 species, but none of the tested birds were positive for M. gallisepticum or M. synoviae. We found possible associations between the presence of Mycoplasma spp. and all investigated ecological factors. The phylogenetic analysis showed a high variability of Mycoplasma spp.; however, some clustering of sequences was observed regarding particular bird species. We found that wild migratory waterfowl, particularly the white-fronted goose (Anser albifrons) and mallard (Anas platyrhynchos) could be reservoirs and vectors of mycoplasmas pathogenic to commercial waterfowl.

Bacterial communities of the upper respiratory tract of turkeys.

The respiratory tracts of turkeys play important roles in the overall health and performance of the birds. Understanding the bacterial communities present in the respiratory tracts of turkeys can be helpful to better understand the interactions between commensal or symbiotic microorganisms and other pathogenic bacteria or viral infections. The aim of this study was the characterization of the bacterial communities of upper respiratory tracks in commercial turkeys using NGS sequencing by the amplification of 16S rRNA gene with primers designed for hypervariable regions V3 and V4 (MiSeq, Illumina). From 10 phyla identified in upper respiratory tract in turkeys, the most dominated phyla were Firmicutes and Proteobacteria. Differences in composition of bacterial diversity were found at the family and genus level. At the genus level, the turkey sequences present in respiratory tract represent 144 established bacteria. Several respiratory pathogens that contribute to the development of infections in the respiratory system of birds were identified, including the presence of Ornithobacterium and Mycoplasma OTUs. These results obtained in this study supply information about bacterial composition and diversity of the turkey upper respiratory tract. Knowledge about bacteria present in the respiratory tract and the roles they can play in infections can be useful in controlling, diagnosing and treating commercial turkey flocks.

Occurrence of Mycoplasma gallisepticum in wild birds: A systematic review and meta-analysis.

Mycoplasma gallisepticum is one of the most important poultry pathogens that can also infect wild birds, but knowledge of potential non-poultry hosts that could be reservoirs of M. gallisepticum is limited. For the paper presented here, we screened three databases (PubMed, Scopus, and the Web of Knowledge) to find articles on the occurrence of M. gallisepticum in different wild bird species that were published between 1951 and 2018. Among 314 studies found, we selected and included 50 original articles that met the pre-established criteria. From those publications we extracted the following information: name of the first author, year of publication, year of sample isolation, country, region, number of birds sampled, number of birds tested by each method, number of positive samples, diagnostic criteria, and if birds were wild or captive. Because different detection techniques were used to confirm the presence of M. gallisepticum in one animal, we decided to perform the meta analyses separately for each method. The estimated prevalence of M. gallisepticum in wild birds was different by each method of detection. Our summary revealed that M. gallisepticum was present in 56 species of bird belonging to 11 different orders, of which 21 species were reported suffering both past and current infection. Our work provides information on wild bird species that could be considered potential reservoirs or carriers of M. gallisepticum and could be helpful to set the direction for future research on the spread and phylogeny of M. gallisepticum in different hosts.

Spectral technique for detection of changes in eggshells caused by Mycoplasma synoviae.

Mycoplasma synoviae (MS) is a major pathogen in chicken and turkeys, causing subclinical infection. MS infections are highly prevalent and may potentate and be involved in sinovitis, respiratory syndromes, as well as lead to eggshell apex abnormality (EAA). A deformed, inhomogeneous eggshell is susceptible to cracks and breaks through which microbes get in and additionally entails higher water loss in the egg during the entire incubation process. Not all eggs with eggshell apex abnormality possess characteristic deformation and that is why some eggs may be incorrectly classified during a visual inspection. To minimize the above risk, the spectral VIS technique and the analysis based on the classification tree method-CTM is proposed. The method makes use of specially defined parameters extracted from the shape of transmittance spectra of eggshells. Directional coefficients of the lines adjusted to the specific ranges of the transmittance spectrum are used in the process of classifying samples as those from MS-carrying hens and from healthy hens. Three CTM-based classifiers were created for a group of white, brown, and mixed shells. After comparing, it can be concluded that the best results were obtained for the group of brown shells (accuracy 88%, specificity 88%, and false negative rate 13%). The authors present a non-invasive spectral method that utilizes eggshells, i.e., the natural waste from chicken farms. The method enables entering data into the classifiers described in the article. The process provides an opportunity to correctly assign, the examined shell to the group of shells with increased risk-with approx. 86% accuracy. This means that, if a few of such results are registered, the herd is eligible more specific studies targeting MS bacteria. Regular spectral testing can support the detection of egg lesions in MS positive flocks.

Prevalence and Phylogenetic Analysis of Mycoplasma Synoviae Strains Isolated from Polish Chicken Layer Flocks.

INTRODUCTION: Mycoplasma synoviae (MS) is a chicken pathogen of major economic importance. MATERIAL AND METHODS: Between 2010 and 2016, 906 commercial layer chicken flocks in Poland were examined for MS, and the phylogenetic relationship among the strains was established. Regionally dispersed samples were collected and tested with the use of real-time PCR to detect the 16S-23S intergenic spacer region. Positive samples were also tested with LAMP and conventional PCR to detect the vlhA gene. RESULTS: MS genetic material was detected in 265 (29%) of the tested flocks by real-time PCR, in 227 by the LAMP method and in 202 (22%) by conventional PCR. The by-year percentage of positive samples began at 34% in 2010, rose to 44% in 2012, and declined to 29% in 2016. A phylogenetic analysis of Polish M. synoviae strains using a partial sequence of the vlhA gene showed nine genotypes (A-I), the most frequently occurring being F and C. Pathogenic Polish MS field isolates (n = 27) collected from chickens with clinical signs of infection were grouped for their characteristic symptoms: respiratory for genotypes C, E, F, and I (n = 13), EAA and a drop in laying for genotypes F, E, and C (n = 12), and synovitis for genotype A (n = 2). CONCLUSION: These data showed the country's isolate diversity. The high prevalence suggests the need to introduce appropriate control programmes. This is the first report of molecular epidemiological data on M. synoviae infection in layer chickens in Poland.

Eggshell apex abnormalities caused by two different Mycoplasma synoviae genotypes and evaluation of eggshell anomalies by full-field optical coherence tomography.

BACKGROUND: Mycoplasma synoviae (MS) is an important poultry pathogen worldwide. This bacterium may cause eggshell changes including an altered shell surface, thinning, and increased translucency in different areas, which leads to a greater incidence of eggshell cracks and breaks. In the present study the association between experimental infection of birds with two field strains of MS from different genotypes and the production of abnormal eggs is described. The analysis of those eggshells using a full-field optical coherence tomography (FF OCT) scanner is also reported. RESULTS: Eggshell samples were obtained from three experimental groups of chickens: one control and two infected tracheally with field strains of MS which produced abnormal eggs. In both experimental groups infected with MS a reduction of mean daily egg production by 11% was observed compared to the control group, which started at 21 to 42 dpi. Eggshell apex abnormalities increased to 24.5% of eggs and in some cases, soft-shelled eggs were produced. This study provides the first analysis of shells from anomalous eggs carried out using FF OCT, which allows three-dimensional structural imaging of an investigated sample at micrometre scale. FF OCT showed ultrastructural changes in eggshells and a smaller number of pores on the entire surface of the affected shells. CONCLUSIONS: The eggshell pathology and the concomitant egg production losses that result from infections highlight the economic significance of MS in commercial poultry. There are differences in the strains of MS which may induce eggshell apex abnormalities (EAA) and egg production losses. The use of FF OCT, which is a noninvasive measurement method based on analysis of the light backscattered from the measured object, will confer the ability to control the quality of eggshells in flocks infected with MS.

Rapid detection of Mycoplasma synoviae by loop-mediated isothermal amplification.

Mycoplasma synoviae (MS) remains a serious concern in production of poultry and affects world production of chickens and turkeys. Loop-mediated isothermal amplification (LAMP) of DNA has been recently used for the identification of different economically important avian pathogens. The aim of this study was to develop LAMP for simple and inexpensive detection of MS strains in poultry using specifically designed primers targeting hemagglutin A (vlh) gene. The assay was conducted in a water bath for 1 h at 63 °C. The results were visualized after addition of SYBR Green(®) fluorescent dye. LAMP was specific exclusively for MS without cross-reactivity with other Mycoplasma species. The sensitivity of LAMP was determined as 10(-1) CFU/ml and was 1,000 times higher than MS-specific polymerase chain reaction. LAMP assay was conducted on 18 MS field strains to ensure its reliability and usefulness. This is the first report on LAMP development and application for the rapid detection of MS isolated from chickens. This simple method may be applied by diagnostic laboratories without access to expensive equipment.