Respiratory Tract Of Chickens Research Articles

Phenotypic and genotypic characterization of extended spectrum beta-lactamase producing E. coli harboring carbapenem and colistin-resistant genes from poultry farms in Egypt.

eEscherichia coli (E. coli) bacteria that produce extended spectrum beta-lactamase (ESBL) is associated with a high prevalence of human illnesses worldwide. The emergence of resistance to carbapenem and colistin compounds poses further challenges to the treatment options for these illnesses. This study aimed to evaluate the phenotypic and genotypic pattern of resistance to carbapenem and colistin in ESBL-producing E. coli. Escherichia coli isolates collected from the respiratory tract of chickens in El-Sharkia government, Egypt. A total of 250 lung samples were collected from 50 poultry farms. These samples were then subjected to isolation, identification, and serotyping of E. coli. The presence of antimicrobial resistance was identified by disc diffusion testing. The occurrence of ESBL phenotypes was also assessed using the double disc synergy method. PCR/sequencing techniques were employed to examine the presence of ESBL (β-lactamase (bla)-TEM, blaSHV, and blaCTX-M), colistin (mcr-1), and carbapenem (blaNDM, blaVIM, and blaKPC) resistance genes. The findings revealed that 140 out of 250 (56%) were identified as E. coli. All E. coli isolates had a high level of multi-antimicrobial resistance (MAR) with an index value greater than 0.2, and 65.7% of them were confirmed to produce ESBL. Out of the 92 ESBL phenotypes, 55 (59.7%), 32 (34.7%), 18 (19.6%), and 37 (40.2%) isolates harbor b laTEM-3, b laSHV-4, b laCTX-M-1, a nd blaCTX-M-14 genes, respectively. The blaNDM-1 gene was identified in all 40 phenotypes that exhibited resistance to carbapenem, accounting for 28.5% of all strains of E. coli and 43.4% of ESBL isolates. The VIM and KPC genes were not detected in any of the samples. Furthermore, there was a significant prevalence of the mobilized colistin resistance (mcr)-1 gene, with 64 (69.5%) of the ESBL isolates exhibiting this gene. The prevalence of ESBL-producing E. coli, particularly those resistant to carbapenem and colistin, poses a significant public health risk in society.

Distribution of importin-α isoforms in poultry species and their tissue- and age-related differences

While importin-α is well studied in mammals, the knowledge in avian species is still limited. In this study, we compared the mRNA expression patterns of five importin-α isoforms in the respiratory tract, liver, and spleen of chickens, turkeys, and pekin ducks in two different age-groups. In addition, we determined the distribution of importin-α in selected tissue of conchae, trachea, and lung of post-hatch chickens at all cellular levels by immunohistochemical staining. Our results indicate that importin-α3 is the most abundant isoform in the respiratory tract of chickens, turkeys, and pekin ducks. Moreover, importin-α is expressed as a gradient with lowest mRNA levels in the conchae and highest levels in the lung. The mRNA expression levels of most isoforms were higher in tissues from post-hatch chickens and turkeys in comparison to the corresponding embryos. In contrast to that, duck embryos mostly show higher mRNA expression levels of importin-α than post-hatch ducks.

Prevalence of multidrug-resistant and extended-spectrum β-lactamase-producing Escherichia coli from chicken farms in Egypt.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains exhibit antibiotic resistance and are known to infect humans worldwide. This study assessed the phenotypic and genotypic prevalence of ESBL-resistant E. coli isolates recovered from the respiratory tracts of chickens in El-Sharkia Governorate, Egypt. We obtained 250 lung samples (one lung/bird) from 50 chicken farms (5 chickens/farm) to isolate, identify, and serotype E. coli. Antimicrobial resistance susceptibility was determined using the disk diffusion method, while the ESBL phenotype was identified using double disk synergy. We detected the β-lactamase genes, blaTEM, and blaSHV, using a polymerase chain reaction. The results showed that 140/250 (56%) were infected with E. coli. All the serogroups of isolated E. coli exhibited high multi-antimicrobial resistance index values (>0.2), and 65.7% were confirmed to have ESBL. Among the isolates with the ESBL phenotypes, 55 (60%) and 32 (35%) contained the blaTEM and blaSHV genes, respectively. The widespread distribution of multidrug-resistant and ESBL-producing E. coli among poultry farms is a significant human health hazard. These results will help the Egyptian authorities to implement a national one-health approach to combat the antimicrobial resistance problem.

New Insights into the Host-Pathogen Interaction of Mycoplasma gallisepticum and Avian Metapneumovirus in Tracheal Organ Cultures of Chicken.

Respiratory pathogens are a health threat for poultry. Co-infections lead to the exacerbation of clinical symptoms and lesions. Mycoplasma gallisepticum (M. gallispeticum) and Avian Metapneumovirus (AMPV) are two avian respiratory pathogens that co-circulate worldwide. The knowledge about the host–pathogen interaction of M. gallispeticum and AMPV in the chicken respiratory tract is limited. We aimed to investigate how co-infections affect the pathogenesis of the respiratory disease and whether the order of invading pathogens leads to changes in host–pathogen interaction. We used chicken tracheal organ cultures (TOC) to investigate pathogen invasion and replication, lesion development, and selected innate immune responses, such as interferon (IFN) α, inducible nitric oxide synthase (iNOS) and IFNλ mRNA expression levels. We performed mono-inoculations (AMPV or M. gallispeticum) or dual-inoculations in two orders with a 24-h interval between the first and second pathogen. Dual-inoculations compared to mono-inoculations resulted in more severe host reactions. Pre-infection with AMPV followed by M. gallispeticum resulted in prolonged viral replication, more significant innate immune responses, and lesions (p < 0.05). AMPV as the secondary pathogen impaired the bacterial attachment process. Consequently, the M. gallispeticum replication was delayed, the innate immune response was less pronounced, and lesions appeared later. Our results suggest a competing process in co-infections and offer new insights in disease processes.

The contribution of the immune response to enhanced colibacillosis upon preceding viral respiratory infection in broiler chicken in a dual infection model

Colibacillosis in chickens caused by avian pathogenic Escherichia coli (APEC) is known to be aggravated by preceding infections with infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and avian metapneumovirus (aMPV). The mechanism behind these virus-induced predispositions for secondary bacterial infections is poorly understood. Here we set out to investigate the immunopathogenesis of enhanced respiratory colibacillosis after preceding infections with these three viruses. Broilers were inoculated intratracheally with APEC six days after oculonasal and intratracheal inoculation with IBV, NDV, aMPV or buffered saline. After euthanasia at 1 and 8 days post infection (dpi) with APEC, birds were macroscopically examined and tissue samples were taken from the trachea, lungs and air sacs. In none of the groups differences in body weight were observed during the course of infection. Macroscopic lesion scoring revealed most severe tissue changes after NDV-APEC and IBV-APEC infection. Histologically, persistent tracheitis was detected in all virus-APEC groups, but not after APEC-only infection. In the lungs, mostly APEC-associated transient pneumonia was observed. Severe and persistent airsacculitis was present after NDV-APEC and IBV-APEC infection. Bacterial antigen was detected by immunohistochemistry only at 1 dpi APEC, predominantly in NDV-APEC- and IBV-APEC-infected lungs. Higher numbers of CD4+ and CD8+ lymphocytes persisted over time in NDV-APEC- and IBV-APEC-infected tracheas, as did CD4+ lymphocytes in NBV-APEC- and IBV-APEC-infected air sacs. KUL01+ cells, which include monocytes and macrophages, and TCRγδ+ lymphocytes were observed mostly in lung tissue in all infected groups with transient higher numbers of KUL01+ cells over time and higher numbers of TCRγδ+ lymphocytes mainly at 8 dpi. qPCR analysis revealed mostly trends of transient higher levels of IL-6 and IFNγ mRNA in lung tissue after IBV-APEC and also NDV-APEC infection and persistent higher levels of IL-6 mRNA after aMPV-APEC infection. In spleens, transient higher levels of IL-17 mRNA and more persistent higher levels of IL-6 mRNA were observed after all co-infections. No changes in IL-10 mRNA expression were seen. These results demonstrate a major impact of dual infections with respiratory viruses and APEC, compared to a single infection with APEC, on the chicken respiratory tract and suggest that immunopathogenesis contributes to lesion persistence.

Detection of Laryngotracheitis Virus in Poultry Flocks with Respiratory Disorders in Slovenia.

Infectious laryngotracheitis (ILT) is an acute, highly contagious infectious disease of the upper respiratory tract in chickens and other poultry species that causes significant economic losses in countries worldwide. Between 2017 and 2019, seven outbreaks of mild to severe respiratory disorders with high suspicion of ILT occurred in commercial and backyard poultry flocks in Slovenia. In all submissions, infection with ILT virus (ILTV) was confirmed by PCR, which is the first report of ILT in Slovenia. Circulating ILT strains were characterized by the sequence and phylogenetic analysis of two fragments of the ICP4 gene. Four strains—three detected in non-vaccinated flocks and one in a flock vaccinated against ILT—were identical or very similar to the chicken embryo–origin live virus vaccines, and the other three were closely related to Russian, Chinese, Australian, and American field strains and to tissue culture origin vaccine strains. As in other diseases, coinfections with other respiratory pathogens in confirmed ILT cases may cause a more severe condition and prolong the course of the disease. In our study, coinfections with Mycoplasma synoviae (7/7 tested flocks), infectious bronchitis virus (5/5 tested flocks), Mycoplasma gallisepticum (4/7 tested flocks), Ornithobacterium rhinotracheale (3/4 tested flocks), and avian pox virus (1/2 tested flocks) were confirmed, indicating the importance of these pathogens in the occurrence of ILT infections.

The prevalence of extended spectrum β–lactamase producing E. coli isolated from chickens in El-sharkia Governorate

E. coli producing extended spectrum beta-lactamase (ESBL) in poultry has a major concern due to the possible transmission between them and human that may cause a public health threat. Our study is directed to estimate the prevalence of resistance pattern and characterization of ESBL producibility and genes in E. coli isolated from chicken respiratory tract from chiken farms in El-Sharkia Governorate. 250 samples from 50 chicken farms were collected. Isolation and identification of E. coli serotypes were performed. In addition, Antibiotic Resistance Profile (ARP) against β-lactam antibiotics, ESBL phenotypic screening and PCR for ESBL genes; SHV and TEM, were done. The results showed that 140 isolates out of 250 total isolates (56%) were morphologically and biochemically positive for E. coli. Different serogroups of isolated E. coli exhibited high resistance rates against 14 antibiotics. 35.7 % of E coli isolates were recorded to be phenotypically ESBL-positive producing bacteria. Genotypically, ESBL genes including bla TEM and bla SHV genes were detected in 50 E. coli samples in (100%) and 30 (60%), respectively. In conclusion, the high prevalence of E. coli producing ESBL genes in poultry farms of El-Sharkia Governorate would account for economic and public health threat in the society.

Research Note: Epidemiological cutoff values and acquired resistance mechanisms of three veterinary antibiotics against Escherichia coli from chicken respiratory tract infections

Florfenicol, apramycin, and danofloxacin are antibiotics approved only for veterinary use and that have good therapeutic effects on chicken respiratory infections caused by Escherichia coli. We established epidemiological cutoff values (ECV) for these antibiotics using 363 E. coli isolates from tracheal samples of chickens in 5 veterinary clinics in Guangdong Province, China. The minimum inhibitory concentrations (MIC) were determined using the agar dilution method as per Clinical and Laboratory Standards Institution guidelines. The ECV were then calculated using the statistical method and verified by normalized resistance interpretation and ECOFFinder software programs. The ECV of florfenicol, apramycin, and danofloxacin against E. coli were 16, 16, and 0.125 μg/mL, respectively. Susceptibility tests indicated that these isolates were resistant to florfenicol (66.7%), apramycin (22.3%), and danofloxacin (92.3%). Strains carrying floR were distributed in the range of MIC ≥32 μg/mL for florfenicol. Apramycin resistance was found in 77 strains (77/363, 21.1%), and isolates that carried aac(3)-IV were all in the range of MIC ≥512 μg/mL. Danofloxacin resistance was found in the range of MIC ≤0.125 μg/mL, but there were no mutations in the quinolone resistance–determining regions and plasmid-mediated quinolone resistance genes qnrA, qnrB, qnrC, qnrD, aac-(6′)-Ib-cr, qep, and oqxB. The presence of the qnrS gene was verified in a few of the strains with an MIC of 0.06 μg/mL. The establishment of ECV was significant for monitoring of resistance development and therapy guidance.

A closed-tube, single-step, real time, reverse transcription-loop-mediated isothermal amplification assay for infectious bronchitis virus detection in chickens

Infectious bronchitis (IB) is a viral infection of the chicken respiratory tract that causes substantial economic burden on the industry. Simple, specific and rapid diagnosis of this disease is critical for the initiation of appropriate control measures. Conventional molecular diagnostic methods require a relatively sophisticated equipment and skilled staff. Here we describe a rapid, simple, semi-quantative, closed-tube, single-step, real-time- reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for IB and compare our assay with quantative, reverse transcription- polymerase chain reaction (RT-qPCR). The limit of detection (LOD) of our RT-LAMP assay is 1 EID50/ ml. Clinical evaluation of samples from diseased chickens with our RT-LAMP showed a very good concordance with RT-qPCR. Our assay enables simple, specific, rapid molecular detection and semi-quantification of the infectious bronchitis virus (IBV) in veterinary diagnostic laboratories. Furthermore, our RT-LAMP detection is carried out in a sealed tube, eliminating the risk of false-positive results in subsequent tests because of any contamination of the work area as in the case of lateral flow strip or gel electrophoresis-based amplicon detection.

Three Amino Acid Changes in Avian Coronavirus Spike Protein Allow Binding to Kidney Tissue.

Infectious bronchitis virus (IBV) infects ciliated epithelial cells in the chicken respiratory tract. While some IBV strains replicate locally, others can disseminate to various organs, including the kidney. Here, we elucidate the determinants for kidney tropism by studying interactions between the receptor-binding domain (RBD) of the viral attachment protein spike from two IBV strains with different tropisms. Recombinantly produced RBDs from the nephropathogenic IBV strain QX and from the nonnephropathogenic strain M41 bound to the epithelial cells of the trachea. In contrast, only QX-RBD binds more extensively to cells of the digestive tract, urogenital tract, and kidneys. While removal of sialic acids from tissues prevented binding of all proteins to all tissues, binding of QX-RBD to trachea and kidney could not be blocked by preincubation with synthetic alpha-2,3-linked sialic acids. The lack of binding of QX-RBD to a previously identified IBV-M41 receptor was confirmed by enzyme-linked immunosorbent assay (ELISA), demonstrating that tissue binding of QX-RBD is dependent on a different sialylated glycan receptor. Using chimeric RBD proteins, we discovered that the region encompassing amino acids 99 to 159 of QX-RBD was required to establish kidney binding. In particular, QX-RBD amino acids 110 to 112 (KIP) were sufficient to render IBV-M41 with the ability to bind to kidney, while the reciprocal mutations in IBV-QX abolished kidney binding completely. Structural analysis of both RBDs suggests that the receptor-binding site for QX is located at a different location on the spike than that of M41.IMPORTANCE Infectious bronchitis virus is the causative agent of infectious bronchitis in chickens. Upon infection of chicken flocks, the poultry industry faces substantial economic losses by diminished egg quality and increased morbidity and mortality of infected animals. While all IBV strains infect the chicken respiratory tract via the ciliated epithelial layer of the trachea, some strains can also replicate in the kidneys, dividing IBV into the following two pathotypes: nonnephropathogenic (example, IBV-M41) and nephropathogenic viruses (including IBV-QX). Here, we set out to identify the determinants for the extended nephropathogenic tropism of IBV-QX. Our data reveal that each pathotype makes use of a different sialylated glycan ligand, with binding sites on opposite sides of the attachment protein. This knowledge should facilitate the design of antivirals to prevent coronavirus infections in the field.

Avian pathogenic Escherichia coli infection of a chicken lung epithelial cell line

Virulent strains of Escherichia coli (Avian Pathogenic E. Coli: APEC) can cause initial infection of the respiratory tract in chickens potentially leading to systemic infection called colibacillosis, which remains a major cause of economic losses in the poultry industry. The role of epithelial lung cells as first targets of APEC and in initiating the innate immune response is unclear and was investigated in this study. APEC was able to adhere and subsequently invade cells from the chicken lung epithelial CLEC213 cell line exhibiting pneumocyte type II-like characteristics. Invasion was confirmed using confocal microscopy after infection with GFP-labelled APEC. Moreover, the infection resulted in a significant increase in IL-8 gene expression, a chemo-attractant of macrophages and heterophils. Gene expression of interferon α and β were not significantly upregulated and chicken Surfactant Protein A, also did not show a significant upregulation on either gene or protein level. The immune response of CLEC213 cells towards APEC was shown to be similar to stimulation with E. coli LPS. These results establish CLEC213 cells as a novel model system for studying bacterial infection of the lung epithelium and show that these cells may play a role in the initial innate response towards bacterial pathogens.

Novel genotype of infectious bronchitis virus isolated in China

Recombination events are known to contribute to the emergence of novel infectious bronchitis virus (IBV) genotypes. In this study, we carried out detailed phylogenetic analysis and sequence comparisons based on 74 complete nucleotide sequences of the IBV S1 gene, including strain I0636/16 and 73 representative sequences from each genotype and lineage. The results showed that strain I0636/16 represented a novel genotype, designated as lineage 1 within genotype VII (GVII-1). Further comparative genomic analysis revealed at least two recombination sites that replaced the spike gene in a lineage 18 within genotype I (GI-18)-like virus with an as-yet-unidentified sequence, likely derived from another IBV strain, resulting a novel serotype with a lower affinity to the respiratory tract in chickens. To the best of our knowledge, this provides the first evidence for recombination leading to replacement of the complete spike gene and the emergence of a novel genotype/serotype with a lower affinity to the respiratory tract in chickens comparing to one of its parental virus ck/CH/LGX/111119. These results emphasize the importance of limiting exposure to novel IBVs that may serve as a source of genetic material for emerging viruses, as well as the importance of IBV surveillance in chicken flocks.

The In Ovo Delivery of CpG Oligonucleotides Protects against Infectious Bronchitis with the Recruitment of Immune Cells into the Respiratory Tract of Chickens.

The in ovo delivery of cytosine-guanosine (CpG) oligodeoxynucleotides (ODNs) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (TLR)21 signaling pathway. Although the delivery of CpG ODNs in ovo at embryo day (ED) 18 has been shown to reduce infectious bronchitis virus (IBV) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered CpG ODNs are capable of protecting chickens against a post-hatch challenge is unknown. Thus, our objectives were to determine the protective effect of the in ovo delivery of CpG ODNs at ED 18 against IBV infection encountered post-hatch and, then, to investigate the mechanisms of protection. We found significantly higher survival rates and reduced IBV infection in the chickens following the pre-treatment of the ED 18 eggs with CpG ODNs. At 3 days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (CD)8α+ and CD4+ T lymphocytes, and an up-regulation of interferon (IFN)-γ mRNA in the respiratory tract of the chickens. Overall, it may be inferred that CpG ODNs, when delivered in ovo, provide protection against IBV infection induced morbidity and mortality with an enhanced immune response.

Visualisation and characterisation of mononuclear phagocytes in the chicken respiratory tract using CSF1R-transgenic chickens

The respiratory tract is a key organ for many avian pathogens as well as a major route for vaccination in the poultry industry. To improve immune responses after vaccination of chickens through increased uptake of vaccines and targeting to antigen presenting cells, a better understanding of the avian respiratory immune system is required. Transgenic MacReporter birds were used expressing a reporter gene (eGFP or mApple) under the control of the CSF1R promoter and enhancer in cells of the mononuclear phagocyte (MNP) lineage to visualize the ontogeny of the lymphoid tissue, macrophages and dendritic cells, in the trachea, lung and air sac of birds from embryonic day 18–63 weeks of age. Small aggregates of CSF1R-transgene+ cells start to form at the openings of the secondary bronchi at 1 week of age, indicative of the early development of the organised bronchus-associated lymphoid tissue. Immunohistochemical staining revealed subpopulations of MNPs in the lung, based on expression of CSF1R-transgene, CD11, TIM4, LAMP1, and MHC II. Specialised epithelial cells or M cells covering the bronchus-associated lymphoid tissue expressed CSF1R-transgene and type II pneumocytes expressed LAMP1 suggesting that these epithelial cells are phagocytic and transcytose antigen. Highly organised lymphoid tissue was seen in trachea from 4 weeks onwards. Throughout the air sacs at all ages, CSF1R-transgene+ cells were scattered and at later stages, CSF1R-transgene+ cells lined capillaries. These results will serve as a base for further functional characterization of macrophages and dendritic cells and their role in respiratory diseases and vaccine responses.

Molecular detection and phylogenetic properties of isolated infectious bronchitis viruses from broilers in Ahvaz, southwest Iran, based on partial sequences of spike gene.

Infectious bronchitis (IB) is a highly contagious disease involving mostly upper respiratory tract in chickens, leading to significant economic losses in the poultry industry worldwide. One of the major concerns regarding to IB is the emergence of new types of infectious bronchitis viruses (IBVs). The purpose of this study was to identify the IBVs isolated from Iranian broiler chickens with respiratory symptoms. Twenty-five broiler flocks around Ahwaz (southwest of Iran) were examined for IBV. The specimens including trachea, lung, liver, kidney, and ceacal tonsil, were collected from diseased birds and inoculated into chicken embryonated eggs. Harvested allantoic fluids were subjected to reverse transcription polymerase chain reaction (RT-PCR) using primers in order to amplify spike 1 (S1) gene of IBV. The RT-PCR products of four IBV isolates were sequenced. The results showed that from 25 examined flocks with respiratory disease, 12 flocks (48.00%) were positive for IBV. In phylogenetic analysis, our isolates were closely related to the QX-like viruses such as PCRLab/06/2012 (Iran), QX, HC9, HC10, CK/CH/GX/NN11-1, CK/CH/JS/YC11-1, CK/CH/JS/2010/13, CK/CH/JS/2011/2 (China), QX/SGK-21, QX/SGK-11 (Iraq) with nucleotide homology up to 99.00%. This study indicates the role of IBVs in the respiratory disorders of broiler flocks located in southwest Iran, and also the existence of a variant of IBV, which is distinguishable from the other Iranian variants.

111 Inhibition of Avian Influenza Virus by Blocking Specific Sialyltransferases

It is known that avian influenza penetrates into the host cell by binding with sialic acids, the terminal residues of oligosaccharides. Avian influenza A virus preferably recognises α(2,3)-linked sialic acid residues as a receptor for penetration whereas human influenza A virus preferably binds with α(2,6)-linked sialic acids. Prevention of transfer of sialic acids to sugar bond or removal of it could be a defensive strategy against viral infection. There are 6 known sialyltransferases (ST3Gal1-6) that transfer α(2,3)-linked sialic acid residues to sugar branches. Most avian influenza virus isolates bind strongly to a sugar chain containing Neu5Aca(2,3) residues. In our study, we have shown that knockout of sialyltransferases leads to inhibition of viral infection. To find the expressed sialyltransferases in respiratory and digestive tracts, we used RT-qPCR. Tissue samples were taken from 3 chickens of Haisex white cross. Expression of mRNA was measured by RT-qPCR in 3 repeats and serial dilutions. Data analysis was carried out using the 2−ΔΔCt method. The amount of total RNA was normalised using GAPDH mRNA. For CRISPR/Cas9 targeting sialyltransferases, 3 guide RNAs for each gene were designed. We confirmed knockout (KO) of ST3GAL1 and ST3GAL6 by T7E assay. To estimate sialylation level on the cell surface, we performed a lectin-binding assay. For the assay, cells were incubated with fluorescein isothiocyanate (FITC)-labelled Maackia amurensis lectins and then subjected to flow-cytometry analysis to quantify the percentage of α(2,3)-sialylated cells in DF1 knockout (KO) v. DF1 wild type (wt) cell line. To estimate resistance to viral infection, a hemagglutinin binding assay was done, using fluorescein isothiocyanate (FITC)-labelled HA1 from H5N1 (A/Vietnam/1203/2004). To quantify the percentage of agglutinated HA1 molecules, DF1 KO and DF1 wt cells were analysed by flow cytometry. We found that mainly ST3GAL4 and ST3GAL5 are expressed in the chicken intestine (3-fold and 20-fold less compared with GAPDH level, respectively; other STs were not detected), and mainly ST3GAL1 and ST3GAL6 are expressed in the chicken respiratory tract (5-fold and 1.2-fold more compared with GAPDH level respectively; other STs were not detected). The expression profile of α(2,3)-sialyltransferases in the DF1 chicken cell line showed the noticeable expression of ST3GAL1 and ST3GAL6 compared with others as has been shown for the respiratory tract (500- and 1000-fold less compared with GAPDH respectively; other STs were not detected). In this study, we adopted the CRISPR/Cas9 system to knock out ST3GAL1 and ST3GAL6 genes in the chicken DF1 cell line. We confirmed that knockout of the genes leads to extinction of α(2,3)-sialic residues from the cell surface (7% v. 100% for DF1 KO v. DF1 wt cell line). Finally, we showed that knockout of sialyltransferases in the DF1 cells increases resistance against influenza A infection (16% v. 100% for DF1 KO v. DF1 wt cell line). Thus, creation of transgenic poultry with tissue-specific knockout of the α(2,3) sialyltransferases might protect domestic birds against influenza virus and block possible transfer of avian flu to human population.

Age-related differences in the respiratory microbiota of chickens.

In this era of next generation sequencing technologies it is now possible to characterise the chicken respiratory microbiota without the biases inherent to traditional culturing techniques. However, little research has been performed in this area. In this study we characterise and compare buccal, nasal and lung microbiota samples from chickens in three different age groups using 16S rRNA gene analysis. Buccal and nasal swabs were taken from birds aged 2 days (n = 5), 3 weeks (n = 5) and 30 months (n = 6). Bronchoalveolar lavage (BAL) samples were also collected alongside reagent only controls. DNA was extracted from these samples and the V2-V3 region of the 16S rRNA gene was amplified and sequenced. Quality control and OTU clustering were performed in mothur. Bacterial DNA was quantified using qPCR, amplifying the V3 region of the 16S rRNA gene. We found significant differences between the quantity and types of bacteria sampled at the three different respiratory sites. We also found significant differences in the composition, richness and diversity of the bacterial communities in buccal, nasal and BAL fluid samples between age groups. We identified several bacteria which had previously been isolated from the chicken respiratory tract in culture based studies, including lactobacilli and staphylococci. However, we also identified bacteria which have not previously been cultured from the respiratory tract of the healthy chicken. We conclude that our study can be used as a baseline that future chicken respiratory microbiota studies can build upon.

Attenuated Phenotype of a Recent House Finch-Associated Mycoplasma gallisepticum Isolate in Domestic Poultry.

Mycoplasma gallisepticum, known primarily as a respiratory pathogen of domestic poultry, has emerged since 1994 as a significant pathogen of the house finch (Haemorhousmexicanus) causing severe conjunctivitis and mortality. House finch-associated M. gallisepticum (HFMG) spread rapidly and increased in virulence for the finch host in the eastern United States. In the current study, we assessed virulence in domestic poultry with two temporally distant, and yet geographically consistent, HFMG isolates which differ in virulence for house finches-Virginia 1994 (VA1994), the index isolate of the epidemic, and Virginia 2013 (VA2013), a recent isolate of increased house finch virulence. Here we report a significant difference between VA1994 and VA2013 in their levels of virulence for chickens; notably, this difference correlated inversely to the difference in their levels of virulence for house finches. VA1994, while moderately virulent in house finches, displayed significant virulence in the chicken respiratory tract. VA2013, while highly virulent in the house finch, was significantly attenuated in chickens relative to VA1994, displaying less-severe pathological lesions in, and reduced bacterial recovery from, the respiratory tract. Overall, these data indicate that a recent isolate of HFMG is greatly attenuated in the chicken host relative to the index isolate, notably demonstrating a virulence phenotype in chickens inversely related to that in the finch host.

Mycoplasma gallisepticum requires ATP to activate the canonical inflammasome in vitro.

Abstract Mycoplasma gallisepticum (MG) is a bacteria which lacks a cells wall and colonizes mucosal epithelial cells. This colonization commonly occurs in the respiratory tracts of chickens and turkeys, leading to chronic respiratory disease. MG is an economically significant pathogen in poultry and can cause large economic losses on poultry farms because of decreased egg laying. Although the importance of MG as an economically relevant pathogen is understood, there is a paucity of knowledge in regards to its pathogenesis. One possible mechanism is via the activation of the inflammasome, a multi-protein complex leading to the maturation of IL-1β and IL-18 as well as the inflammatory form of cell death called pyroptosis. Interestingly, Mycoplasma hyorhinis (MH) was shown to activate the NLRP3 inflammasome in THP-1 and mouse bone marrow derived dendritic cells (BMDCs). In addition, HD-11 cells co-cultured with tracheal epithelial cells that were exposed to MG, show increased levels of IL-1β. In this study, we aimed to determine if MG activated the inflammasome in murine and chicken macrophages. We found that MG activates the canonical inflammasome in primary murine macrophages via the induction of IL-1b release. However, we determined that this MG-mediated activation of the canonical inflammasome requires the addition of ATP in vitro. In addition, we also found that MG alone is not sufficient to activate pyroptosis in vitro. Interestingly, MG also initiated the production of IL-6 in these cells, suggesting potential activation of the NF-κB pathway. We are currently working with the HD-11 chicken cell line to determine if MG activates the inflammasome in these cells as well.

Immunohistochemical and clinical changes in the respiratory tract of chickens, naturally infected with the fowlpox virus

It is clear that currently the question of study of the pathological changes in the respiratory tract of chickens due to the impact of the fowlpox virus remains relevant, as the pathogenesis of nutritional deficiency, the presence of mycotoxins or the papilloma virus are characterized by the same clinical manifestations. We analyzed 88 chickens, naturally infected with the fowlpox virus, using clinical and immunohistochemical methods of investigation. Among all species of birds, we studied only chickens, data on which were collected for five years from veterinary clinics. The investigation revealed lesions basically characterized by the presence of changes in the typical structure of the chicken’s respiratory tract. As a result, we found clear criteria for the pathological process in the respiratory tract of chickens, which are typical for fowlpox. Thus, we discovered respiratory tract obstruction, as well as many intracytoplasmic pale eosinophilic inclusions in hyperplastic cells. We found an accumulation of mononuclear cells consisting mainly of macrophages, lymphocytes, plasma and mononuclear cells inside the mucous and muscle membranes. Bronchial lumens were blocked by necrotic and desquamated epithelial cells, red blood cells, bacterial colonies and amorphous eosinophilic material. We found accumulations of lymphocytes and macrophages in the parenchyma of the lungs. The hyperplastic epithelial cells reacted immunohistochemically with antibodies against the fowlpox virus in the respiratory tract. Immunoreaction occurred mainly in the cytoplasm of infected cells, inclusions, and necrotic and desquamated cells. The study proved immunohistochemical methods of investigation can be a useful additional tool for establishing a final diagnosis, especially in acute and subacute phases of the disease. The following respiratory signs were observed in severe cases of fowlpox: damage to the lungs in 33 cases (46.5%), parabronchium – 20 (28.2%), parabronchial connective tissue – 8 (11.3%), and mucous membrane of the larynx and trachea – 10 (14.1%). In mild and moderate cases of fowlpox, the following respiratory signs were observed: hyperemia and thickening of the mucous membrane of the trachea – 14 (82.4%), as well as hyperemia in the nasal conchae and paranasal sinuses – 3 (17.7%). In the future, it will be necessary to conduct deeper studies to detect pathological manifestations of this disease, not only in the respiratory tract, but also in other organs and systems of chickens.