The GE296_RS03820 and GE296_RS03830 genes are involved in capsular polysaccharide biosynthesis in Riemerella anatipestifer.

Riemerella anatipestifer is a pathogenic bacterium that causes duck serositis and meningitis, leading to significant harm to the duck industry. To escape from the host immune system, the meningitis-causing bacteria must survive and multiply in the bloodstream, relying on specific virulence factors such as capsules. Therefore, it is essential to study the genes involved in capsule biosynthesis in R. anatipestifer. In this study, we successfully constructed gene deletion mutants Δ3820 and Δ3830, targeting the GE296_RS03820 and GE296_RS03830 genes, respectively, using the RA-LZ01 strain as the parental strain. The growth kinetics analysis revealed that these two genes contribute to bacterial growth. Transmission and scanning electron microscopy (TEM and SEM) and silver staining showed that Δ3820 and Δ3830 produced the altered capsules and compounds of capsular polysaccharides (CPSs). Serum resistance test showed the mutants also exhibited reduced C3b deposition and decreased resistance serum killing. In vivo, Δ3820 and Δ3830 exhibited markedly declining capacity to cross the blood-brain barrier, compared to RA-LZ01. These findings indicate that the GE296_RS03820 and GE296_RS03830 genes are involved in CPSs biosynthesis and play a key role in the pathogenicity of R. anatipestifer. Furthermore, Δ3820 and Δ3830 mutants presented a tendency toward higher survival rates from RA-LZ01 challenge in vivo. Additionally, sera from ducklings immunized with the mutants showed cross-immunoreactivity with different serotypes of R. anatipestifer, including 1, 2, 7 and 10. Western blot and SDS-PAGE assays revealed that the altered CPSs of Δ3820 and Δ3830 resulted in the exposure of some conserved proteins playing the key role in the cross-immunoreactivity. Our study clearly demonstrated that the GE296_RS03820 and GE296_RS03830 genes are involved in CPS biosynthesis in R. anatipestifer and the capsule is a target for attenuation in vaccine development.

The First Case of Menacanthus pallidulus (Neumann, 1912) (Phthiraptera: Amblycera: Menoponidae) on A Chicken (Gallus gallus domesticus Linnaeus, 1758) in Türkiye.

This case report was prepared to provide information about Menacanthus pallidulus (Neumann, 1912), which was detected for the first time on a domestic chicken in Hatay province of Türkiye. Louse specimens collected from a chicken by a student were brought to Hatay Mustafa Kemal University Faculty of Veterinary Medicine, Department of Parasitology, and sent to Selçuk University Faculty of Veterinary Medicine, Department of Parasitology, for identification of species and microscopic examination revealed the presence of Menacanthus pallidulus (Neumann, 1912). Thus, with this study, the presence of M. pallidulus on domestic chickens was recorded for the first time in Türkiye.

Insights of early feeding regime supplemented with glutamine and various levels of omega-3 in broiler chickens: growth performance, muscle building, antioxidant capacity, intestinal barriers health and defense against mixed Eimeria spp infection.

Early nutritional management approach greatly impacts broilers' performance and resistance against coccidiosis. The current study explored the impact of post-hatch feeding with a combination of glutamine (Glut) and different levels of omega-3 on broiler chickens' growth performance, muscle building, intestinal barrier, antioxidant ability and protection against avian coccidiosis. A total of six hundred Cobb 500 was divided into six groups: first group (fed basal diet and unchallenged (control) and challenged (negative control, NC) groups were fed a basal diet without additives, and the other groups were infected with Eimeria spp and supplemented with 1.5% Glut alone or with three different levels of omega-3 (0.25, 0.5 and 1%) during the starter period. Notable improvement in body weight gain was observed in the group which fed basal diet supplemented with glut and 1% omega 3 even after coccidia infection (increased by 25% compared challenged group) while feed conversion ratio was restored to control. Myogeneis was enhanced in the group supplemented with Glut and omega-3 (upregulation of myogenin, MyoD, mechanistic target of rapamycin kinase and insulin like growth factor-1 and downregulating of myostatin genes). Groups supplemented with Glut and higher levels of omega-3 highly expressed occluding, mucin-2, junctional Adhesion Molecule 2, b-defensin-1 and cathelicidins-2 genes. Group fed 1% Glut + omega-3 showed an increased total antioxidant capacity and glutathione peroxidase and super oxide dismutase enzymes activities with reduced levels of malondialdehyde, reactive oxygen species and H2O2. Post-infection, dietary Glut and 1% omega-3 increased intestinal interleukin-10 (IL) and secretory immunoglobulin-A and serum lysozyme, while decreased the elevated inflammatory mediators comprising interleukin IL-6, tumor necrosis factor-alpha, nitric oxide (NO) and inducible NO synthase. Fecal oocyst excretion and lesions score severity were lowered in the group fed 1% Glut and omega 3. Based on these findings, dietary Glut and omega-3 supplementation augmented restored overall broilers' performance after coccidial challenge.

Polymeric nanocarrier-based adjuvants to enhance a locally produced mucosal coryza vaccine in chicken.

Infectious coryza (IC) is an acute upper respiratory disease of chicken caused by Avibacterium (A.) paragallinarum. This disease results in an increased culling rate in meat chicken and a marked decrease in egg production (10% to more than 40%) in laying and breeding hens. Vaccines were first used against IC and effectively controlled the disease. Nanotechnology provides an excellent way to develop a new generation of vaccines. NPs have been widely used in vaccine design as adjuvants and antigen delivery vehicles and as antibacterial agents; thus, they can be used as inactivators for bacterial culture. In this research, the antibacterial effects of several nanoparticles (NPs), such as silicon dioxide with chitosan (SiO2-CS), oleoyl-chitosan (O.CS), silicon dioxide (SiO2), and iron oxide (Fe3O4), on A. paragallinarum were studied. Additionally, different A. paragallinarum vaccines were made using the same nanomaterials at a concentration of 400 µg/ml to help control infectious coryza disease in chicken. A concentration of 400 µg/ml of all the NPs tested was the best concentration for the inactivation of A. paragallinarum. Additionally, this study showed that the infectious coryza vaccine adjuvanted with SiO2 NPs had the highest immune response, followed by the infectious coryza vaccine adjuvanted with Fe3O4 NPs, the infectious coryza vaccine adjuvanted with SiO2-CS NPs, and the infectious coryza vaccine adjuvanted with O.CS NPs in comparison with the infectious coryza vaccine adjuvanted with liquid paraffin (a commercial vaccine).

Phenotypic characterization and genomic analysis of a Salmonella phage L223 for biocontrol of Salmonella spp. in poultry.

The escalating incidence of foodborne salmonellosis poses a significant global threat to food safety and public health. As antibiotic resistance in Salmonella continues to rise, there is growing interest in bacteriophages as potential alternatives. In this study, we isolated, characterized, and evaluated the biocontrol efficacy of lytic phage L223 in chicken meat. Phage L223 demonstrated robust stability across a broad range of temperatures (20-70 °C) and pH levels (2-11) and exhibited a restricted host range targeting Salmonella spp., notably Salmonella Typhimurium and Salmonella Enteritidis. Characterization of L223 revealed a short latent period of 30 min and a substantial burst size of 515 PFU/cell. Genomic analysis classified L223 within the Caudoviricetes class, Guernseyvirinae subfamily and Jerseyvirus genus, with a dsDNA genome size of 44,321 bp and 47.9% GC content, featuring 72 coding sequences devoid of antimicrobial resistance, virulence factors, toxins, and tRNA genes. Application of L223 significantly (p < 0.005) reduced Salmonella Typhimurium ATCC 14,028 counts by 1.24, 2.17, and 1.55 log CFU/piece after 2, 4, and 6 h of incubation, respectively, in experimentally contaminated chicken breast samples. These findings highlight the potential of Salmonella phage L223 as a promising biocontrol agent for mitigating Salmonella contamination in food products, emphasizing its relevance for enhancing food safety protocols.

Prevalence and antimicrobial resistance of salmonellae isolated from eggs of local chicken in selected towns of Ethiopia.

BACKGROUND: Salmonellosis is one of the most common food-borne diseases in industrialised and developing countries. In recent year, an increase in antimicrobial resistance among different Salmonella serotypes has been observed. OBJECTIVE: A cross-sectional study was conducted to assess the prevalence and antimicrobial susceptibility of Salmonella isolated from local chicken eggs in four selected towns in Ethiopia. METHODS: A total of 115 eggs were examined to detect Salmonella by using standard microbiological methods. The susceptibilities of the isolates to nine antimicrobials were tested by the Kirby-Bauer disk diffusion method. RESULT: The study revealed that of the 115 eggs examined, 22 (19.1%) were positive for Salmonella of which 14 (12.2%) and 8 (7%) of the isolates were from shells and contents, respectively. The occurrence of Salmonella in egg shells and content and between different altitudes did not differ significantly (p > 0.05). Most isolates were resistant to more than three antimicrobials with a high resistance to kanamycin, ampicillin, nalidixic acid, cotrimoxazole, oxytetracycline and chloramphenicol. CONCLUSION: The results indicate the potential importance of local chicken eggs as source of multiple antimicrobial-resistant salmonellae and the need for proper cooking before consumption. Further studies are required to describe the epidemiology of Salmonella in various agroclimatic zones of Ethiopia.

Morphomolecular identification of heavy parasitic typhlitis in layer flocks: tissue response and cell-mediated reaction.

BACKGROUND: Heterakis gallinarum (H. gallinarum) is a common poultry parasite that can be found in the ceca of many gallinaceous bird species, causing minor pathology and reduced weight gain. Most infections go unnoticed in commercial flocks due to the dependence on fecal egg counts, which are prone to false-negative diagnoses. Furthermore, there is a lack of research on gastrointestinal nematodes that use molecular identification methods, which could be essential for rapid diagnosis and developing efficient control approaches. As a result, the study aimed to look at the cause of mortality in layer chickens induced by H. gallinarum in Egyptian poultry farms using morphological, ultrastructural, and molecular characterization. Histopathological, immunohistochemical, and cell-mediated immune responses from damaged cecal tissues were also examined. RESULTS: Seventy bird samples from ten-layer flocks of different breeds (Native, white, and brown layers) suffering from diarrhea, decreased egg output, and emaciation were collected. Cecal samples were collected from affected and non-affected birds and were examined for parasitic diseases using light and a scanning electron microscope. The mitochondrial cytochrome oxidase 1 (COX1) gene was used to characterize H. gallinarum. Our results showed that the collected nematodal worms were identified as H. gallinarum (male and female), further confirmed by COX1 gene amplification and sequence alignment. Gene expression analysis of the inflammatory markers in infected tissues showed a significant up-regulation of IL-2, IFN-γ, TLR-4, and IL-1ß and a significant down-regulation of the anti-inflammatory IL-10. The mRNA level of the apoptotic cas-3 revealed apoptotic activity among the H. gallinarum samples compared to the control group. CONCLUSIONS: Our results implemented the use of molecular methods for the diagnosis of Heterakis, and this is the first report showing the tissue immune response following infection in layers: upregulation of IL-1ß, IFN-γ, Il-2, and TLR-4, while down-regulation of anti-inflammatory IL-10 in cecal tissue, Cas-3 apoptotic activity and Nuclear factor-κB (NF-κB)activity with immunophenotyping of T-cells in Heterakis infected tissue.

Recombination of variable and host range regions of glycoprotein gp85 in different avian leukosis virus subgroup K isolates.

Given the high prevalence of avian leukosis virus subgroup K (ALV-K) in chickens in China, the positive rate of ALV-K in local chickens in Henan province was investigated, and the genetic region encoding the glycoprotein gp85 of isolates from positive chickens was analyzed. The positive rate of ALV-K in local chickens in Henan was found to be 87.2% (41/47). Phylogenetic analysis of gp85 sequences revealed six clusters that differed in their host range regions (hr1 and hr2) and variable regions (vr1, vr2, and vr3). Evidence of recombination of hr1, hr2, vr1, vr2, and vr3 was observed between the different clusters. The isolate HN23LS02 appears to have obtained its hr1 and hr2 regions from separate lineages via recombination but without having a significant affect on the replication capacity of the virus.

Comparative genomics of quinolone-resistant Escherichia coli from broilers and humans in Norway.

BACKGROUND: The usage of fluoroquinolones in Norwegian livestock production is very low, including in broiler production. Historically, quinolone-resistant Escherichia coli (QREC) isolated from Norwegian production animals rarely occur. However, with the introduction of a selective screening method for QREC in the Norwegian monitoring programme for antimicrobial resistance in the veterinary sector in 2014; 89.5% of broiler caecal samples and 70.7% of broiler meat samples were positive. This triggered the concern if there could be possible links between broiler and human reservoirs of QREC. We are addressing this by characterizing genomes of QREC from humans (healthy carriers and patients) and broiler isolates (meat and caecum). RESULTS: The most frequent mechanism for quinolone resistance in both broiler and human E. coli isolates were mutations in the chromosomally located gyrA and parC genes, although plasmid mediated quinolone resistance (PMQR) was also identified. There was some relatedness of the isolates within human and broiler groups, but little between these two groups. Further, some overlap was seen for isolates with the same sequence type isolated from broiler and humans, but overall, the SNP distance was high. CONCLUSION: Based on data from this study, QREC from broiler makes a limited contribution to the incidence of QREC in humans in Norway.

Oral vaccination with a recombinant Lactobacillus plantarum expressing the Eimeria tenella rhoptry neck 2 protein elicits protective immunity in broiler chickens infected with Eimeria tenella.

BACKGROUND: Chicken coccidiosis is a protozoan disease that leads to considerable economic losses in the poultry industry. Live oocyst vaccination is currently the most effective measure for the prevention of coccidiosis. However, it provides limited protection with several drawbacks, such as poor immunological protection and potential reversion to virulence. Therefore, the development of effective and safe vaccines against chicken coccidiosis is still urgently needed. METHODS: In this study, a novel oral vaccine against Eimeria tenella was developed by constructing a recombinant Lactobacillus plantarum (NC8) strain expressing the E. tenella RON2 protein. We administered recombinant L. plantarum orally at 3, 4 and 5 days of age and again at 17, 18 and 19 days of age. Meanwhile, each chick in the commercial vaccine group was immunized with 3 × 102 live oocysts of coccidia. A total of 5 × 104 sporulated oocysts of E. tenella were inoculated in each chicken at 30 days. Then, the immunoprotection effect was evaluated after E. tenella infection. RESULTS: The results showed that the proportion of CD4+ and CD8+ T cells, the proliferative ability of spleen lymphocytes, inflammatory cytokine levels and specific antibody titers of chicks immunized with recombinant L. plantarum were significantly increased (P < 0.05). The relative body weight gains were increased and the number of oocysts per gram (OPG) was decreased after E. tenella challenge. Moreover, the lesion scores and histopathological cecum sections showed that recombinant L. plantarum can significantly relieve pathological damage in the cecum. The ACI was 170.89 in the recombinant L. plantarum group, which was higher than the 150.14 in the commercial vaccine group. CONCLUSIONS: These above results indicate that L. plantarum expressing RON2 improved humoral and cellular immunity and enhanced immunoprotection against E. tenella. The protective efficacy was superior to that of vaccination with the commercial live oocyst vaccine. This study suggests that recombinant L. plantarum expressing the RON2 protein provides a promising strategy for vaccine development against coccidiosis.

The Effects of Aflatoxin B1 on Liver Cholestasis and Its Nutritional Regulation in Ducks.

The aim of this study was to investigate the effects of aflatoxin B1 (AFB1) on cholestasis in duck liver and its nutritional regulation. Three hundred sixty 1-day-old ducks were randomly divided into six groups and fed for 4 weeks. The control group was fed a basic diet, while the experimental group diet contained 90 µg/kg of AFB1. Cholestyramine, atorvastatin calcium, taurine, and emodin were added to the diets of four experimental groups. The results show that in the AFB1 group, the growth properties, total bile acid (TBA) serum levels and total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and glutathione (GSH) liver levels decreased, while the malondialdehyde (MDA) and TBA liver levels increased (p < 0.05). Moreover, AFB1 caused cholestasis. Cholestyramine, atorvastatin calcium, taurine, and emodin could reduce the TBA serum and liver levels (p < 0.05), alleviating the symptoms of cholestasis. The qPCR results show that AFB1 upregulated cytochrome P450 family 7 subfamily A member 1 (CYP7A1) and cytochrome P450 family 8 subfamily B member 1 (CYP8B1) gene expression and downregulated ATP binding cassette subfamily B member 11 (BSEP) gene expression in the liver, and taurine and emodin downregulated CYP7A1 and CYP8B1 gene expression (p < 0.05). In summary, AFB1 negatively affects health and alters the expression of genes related to liver bile acid metabolism, leading to cholestasis. Cholestyramine, atorvastatin calcium, taurine, and emodin can alleviate AFB1-induced cholestasis.

Postbiotic-based recombinant receptor activator of NF-κB ligand enhanced oral vaccine efficiency in chicken.

Functional M cells are differentiated by receptor activator of NF-κB ligand (RANKL) and capture of luminal antigens to initiate immune responses. We aimed to use postbiotic-based recombinant chicken RANKL (cRANKL) to promote M cell differentiation and test the efficacy of oral vaccines. Chicks were divided into three groups that were administered phosphate-buffered saline (PBS), cell extracts of wild-type Lactococcus lactis subsp. lactis IL1403 (WT_CE), or cell extracts of recombinant L. lactis expressing cRANKL (cRANKL_CE). The expression of the M cell marker was measured, and the gut microbiome was profiled. The efficiency of the infectious bursal disease (IBD) vaccine was tested after 12 consecutive days of administering cRANKL_CE. The chickens that were administered cRANKL_CE (p = 0.038) had significantly higher Annexin A5 (ANXA5) mRNA expression levels than those in the PBS group (PBS vs. WT_CE, p = 0.657). In the gut microbiome analysis, no significant changes were observed. However, the relative abundance of Escherichia-Shigella was negatively correlated (r = - 0.43, p = 0.019) with ANXA5 mRNA expression in Peyer's patches. cRANKL_CE/IBD (p = 0.018) had significantly higher IBD-specific faecal IgA levels than PBS/IBD (PBS/IBD vs. WT_CE/IBD, p = 0.217). Postbiotic-based recombinant cRANKL effectively improved the expression of M cell markers and the efficiency of oral vaccines. No significant changes were observed in the gut microbiome after administration of postbiotic-based recombinant cRANKL. This strategy can be used for the development of feed additives and adjuvants. KEY POINTS: • Postbiotic-based recombinant cRANKL enhanced the expression of ANXA5 in chicken. • The relative abundance of Escherichia-Shigella was negatively correlated with ANXA5 expression. • Postbiotic-based recombinant cRANKL effectively improved the efficiency of oral vaccine.

Recombination and amino acid point mutations in VP3 exhibit a synergistic effect on increased virulence of rMDPV.

Recombinant Muscovy duck parvovirus (rMDPV) is a product of genetic recombination between classical Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV). The recombination event took place within a 1.1-kb DNA segment located in the middle of the VP3 gene, and a 187-bp sequence extending from the P9 promoter to the 5' initiation region of the Rep1 ORF. This resulted in the alteration of five amino acids within VP3. Despite these genetic changes, the precise influence of recombination and amino acid mutations on the pathogenicity of rMDPV remains ambiguous. In this study, based on the rMDPV strain ZW and the classical MDPV strain YY, three chimeric viruses (rZW-mP9, rZW-mPR187, and rYY-rVP3) and the five amino acid mutations-introduced mutants (rZW-g5aa and rYY-5aa(ZW)) were generated using reverse genetic technology. When compared to the parental virus rZW, rZW-g5aa exhibited a prolonged mean death time (MDT) and a decreased median lethal dose (ELD50) in embryonated duck eggs. In contrast, rYY-5aa(ZW) did not display significant differences in MDT and ELD50 compared to rYY. In 2-day-old Muscovy ducklings, infection with rZW-g5aa and rYY-5aa(ZW) resulted in mortality rates of only 20% and 10%, respectively, while infections with the three chimeric viruses (rZW-mP9, rZW-mPR187, rYY-rVP3) and rZW still led to 100% mortality. Notably, rYY-rVP3, containing the VP3 region from strain ZW, exhibited 50% mortality in 6-day-old Muscovy ducklings and demonstrated significant horizontal transmission. Collectively, our findings indicate that recombination and consequent amino acid changes in VP3 have a synergistic impact on the heightened virulence of rMDPV in Muscovy ducklings.

Hemagglutinin glycosylation pattern-specific effects: implications for the fitness of H9.4.2.5-branched H9N2 avian influenza viruses.

Since 2007, h9.4.2.5 has emerged as the most predominant branch of H9N2 avian influenza viruses (AIVs) that affects the majority of the global poultry population. The spread of this viral branch in vaccinated chicken flocks has not been considerably curbed despite numerous efforts. The evolutionary fitness of h9.4.2.5-branched AIVs must consequently be taken into consideration. The glycosylation modifications of hemagglutinin (HA) play a pivotal role in regulating the balance between receptor affinity and immune evasion for influenza viruses. Sequence alignment showed that five major HA glycosylation patterns have evolved over time in h9.4.2.5-branched AIVs. Here, we compared the adaptive phenotypes of five virus mutants with different HA glycosylation patterns. According to the results, the mutant with 6 N-linked glycans displayed the best acid and thermal stability and a better capacity for multiplication, although having a relatively lower receptor affinity than 7 glycans. The antigenic profile between the five mutants revealed a distinct antigenic distance, indicating that variations in glycosylation level have an impact on antigenic drift. These findings suggest that changes in the number of glycans on HA can not only modulate the receptor affinity and antigenicity of H9N2 AIVs, but also affect their stability and multiplication. These adaptive phenotypes may underlie the biological basis for the dominant strain switchover of h9.4.2.5-branched AIVs. Overall, our study provides a systematic insight into how changes in HA glycosylation patterns regulate the evolutionary fitness and epidemiological dominance drift of h9.4.2.5-branched H9N2 AIVs, which will be of great benefit for the glycosylation-dependent vaccine design.

Rapid detection of pigeon adenovirus 2 using a TaqMan real-time PCR assay.

Pigeons infected with aviadenoviruses have been found worldwide. Recently, pigeon adenovirus 2 (PiAdV-2) has been widely distributed in racing pigeons in Germany. However, the epidemiology of this virus remains unclear due to the lack of a specific detection platform for PiAdV-2. In this study, we first detected PiAdV-2 positivity in racing pigeons (designated FJ21125 and FJ21128, which share 100% nucleotide identity with each other based on the fiber 2 gene) in Fujian, Southeast China. These genes shared 99.8% nucleotide identity with PiAdV-2 (GenBank No. NC_031501) but only 54.1% nucleotide identity with PiAdV-1 (GenBank No. NC024474). Then, the TaqMan-qPCR assay for the detection of PiAdV-2 was established based on fiber 2 gene characterization. The established assay had a correlation coefficient of 1.00, with an amplification efficiency of 99.0%. The minimum detection limit was 34.6 copies/µL. Only PiAdV-2 exhibited a positive fluorescent signal, and no signal was detected for other pathogens (including PiCV, FAdV-4, FAdV-8a, EDSV, PPMV-1, RVA and PiHV). The assay has good reproducibility, with a coefficient of variation less than 2.42% both intragroup and intergroup. The distributions of PiAdV-2 in fecal samples from YPDS (35 samples) and healthy (43 samples) racing pigeons from different geographical areas were investigated and were 37.14% (YPDS) and 20.93% (healthy), respectively. In summary, we developed a TaqMan-qPCR platform for the detection of PiAdV-2 infection with high sensitivity, specificity, and reproducibility. We confirmed the presence of PiAdV-2 in China, and our data suggested that there is no indication of a correlation between YPDS and PiAdV-2. This study provides more information on the pathogenesis mechanism and epidemiological surveillance of PiAdV-2.

Evaluating the effects of virulence genotype, swarming motility, and multi-locus sequence types of Escherichia coli on layer chicken embryos.

AIMS: To determine the effects of swarming motility (SM) and multi-locus sequence types (MLST) on the main effect of virulence genotype of Escherichia coli through an embryos lethality assay between the 12th and 18th days of incubation. METHODS AND RESULTS: We collected 58 E. coli isolates from asymptomatic commercial hens (n = 42) and lesions of colibacillosis cases (n = 16), then classified their virulence genotype as avirulent, moderately virulent, virulent-healthy, and virulent-colibacillosis categories by the presence of five virulence-associated genes (iroN, ompT, hlyF, iutA, and iss). These isolates were further classified as non-motile, motile, or hyper-motile by SM assay. From the 58 isolates, we selected 29 for ELA and determined their MLST. Each isolate was inoculated into 15 embryonated eggs through the allantoic cavity. We found the avirulent isolates reduced the relative embryo weight compared to virulent-colibacillosis and moderately virulent isolates (37.49 vs. 41.51 and 40.34%, P = 0.03). Among the moderately virulent and virulent-colibacillosis categories, embryo lethality was lower when isolates were non-motile. Yolk retention was unaffected by virulence categories, motility, or MLST. CONCLUSION: Interaction between virulence genotype and SM substantially influenced the embryo lethality assay of E. coli isolates.

Reversal of gentamicin sulfate resistance in avian pathogenic Escherichia coli by matrine combined with berberine hydrochloride.

In recent years, the evolution of antibiotic resistance has led to the inefficacy of several antibiotics, and the reverse of resistance was a novel method to solve this problem. We previously demonstrated that matrine (Mat) and berberine hydrochloride (Ber) had a synergistic effect against multidrug-resistant Escherichia coli (MDREC). This study aimed to demonstrate the effect of Mat combined with Ber in reversing the resistance of MDREC. The MDREC was sequenced passaged in the presence of Mat, Ber, and a combination of Mat and Ber, which did not affect its growth. The reverse rate was up to 39.67% after MDREC exposed to Mat + Ber for 15 days. The strain that reversed resistance was named drug resistance reversed E. coli (DRREC) and its resistance to ampicillin, streptomycin, gentamicin, and tetracycline was reversed. The MIC of Gentamicin Sulfate (GS) against DRREC decreased 128-fold to 0.63 µg/mL, and it was stable within 20 generations. Furthermore, the susceptible phenotype of DRREC remained stable within 20 generations, as well. The LD50 of DRREC for chickens was 8.69 × 109 CFU/mL. qRT-PCR assays revealed that the transcript levels of antibiotic-resistant genes and virulence genes in the DRREC strain were significantly lower than that in the MDREC strain (P < 0.05). In addition, GS decreased the death, decreased the bacterial loading in organs, alleviated the injury of the spleen and liver, and decreased the cytokine levels in the chickens infected by the DRREC strain. In contrast, the therapeutic effect of GS in chickens infected with MDREC was not as evident. These findings suggest that the combination of Mat and Ber has potential for reversing resistance to MDREC.

Chicken intestinal organoids: a novel method to measure the mode of action of feed additives.

There is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or "mini-guts" has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challenge model. To mimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EO for 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signaling. In contrast, when the organoids were treated with OA+EO and challenged with Salmonella, the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses.