Allantoic Cavity Research Articles

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

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. 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 (37.49vs. 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. Interaction between virulence genotype and SM substantially influenced the embryo lethality assay of E. coli isolates.

Quercetin attenuates lipopolysaccharide-induced hepatic inflammation by modulating autophagy and necroptosis

Lipopolysaccharide (LPS) from Gram-negative bacteria initially induces liver inflammation with proinflammatory cytokines expressions. However, the underlying hepatoprotective mechanism of quercetin on LPS-induced hepatic inflammation remains unclear. Specific pathogen-free chicken embryos (n = 120) were allocated control vehicle, PBS with or without ethanol vehicle, LPS (125 ng/egg) with or without quercetin treatment (10, 20, or 40 nmol/egg, respectively), quercetin groups (10, 20, or 40 nmol/egg). Fifteen-day-old embryonated eggs were inoculated abovementioned solutions via the allantoic cavity. At embryonic d 19, the livers of the embryos were collected for histopathological examination, RNA extraction, real-time polymerase chain reaction, and immunohistochemistry investigation. We found that the liver presented inflammatory response (heterophils infiltration) after LPS induction. The LPS-induced mRNA expressions of inflammation-related factors (TLR4, TNFα, IL-1β, IL-10, IL-6, MYD88, NF-κB1, p38, and MMP3) were upregulated after LPS induction when compared with the PBS group, while quercetin could downregulate these expressions as compared with the LPS group. Quercetin significantly decreased the immunopositivity to TLR4 and MMP3 in the treatment group when compared with the LPS group. Quercetin could significantly downregulate the mRNA expressions of autophagy-related genes (ATG5, ATG7, Beclin-1, LC3A, and LC3B) and necroptosis-related genes (Fas, Bcl-2, Drp1, and RIPK1) after LPS induction. Quercetin significantly decreased the immunopositivity to LC3 in the treatment group when compared with the LPS group; meanwhile, quercetin significantly decreased the protein expressions of LC3-I, LC3-II, and the rate of LC3-II/LC3-I. In conclusions, quercetin can alleviate hepatic inflammation induced by LPS through modulating autophagy and necroptosis.

Genomic characterization and pathogenicity of a novel fowl adenovirus serotype 11 isolated from chickens with inclusion body hepatitis in China

Fowl adenovirus serotype 11 (FAdV-11) is one of the primary causative agents of inclusion body hepatitis (IBH), which causes substantial economic losses in the world poultry industry. In this study, we characterized the genome of the fowl adenovirus serotype 11 (FAdV-11) isolate FJSW/2021. The full genome of FJSW/2021 was 44, 154 base pairs (bp) in length and had a similar organization to that of previously reported FAdV-11 isolates. Notably, compared with those of other reported FAdV-11 strains, the preterminal protein (pTP) of FAdV-11 FJSW/2021 has six amino acid (aa) insertions (S-L-R-I-I-C) between 470 and 475 and one aa mutation of L476F; moreover, the tandem repeat (TR) regions of TR1 and TR2 were 33 bp (1 repeat) and 1,080 bp (8 repeats) shorter than those of the Canadian nonpathogenic isolate ON NP2, respectively. The pathogenicity of FJSW/2021 was studied in 10-day-old specific pathogen-free chicken embryos following allantoic cavity inoculation and in 1-day-old, 1-wk-old and 2-wk-old SPF chickens following intramuscular inoculation with 107 TCID50 of the virus. The results showed that FJSW/2021 can induce typical severe IBH in chicks less than 2 wk old. These findings highlighted the genetic differences between the pathogenic and non-pathogenic FAdV-11 isolates. The data will provide guidance for identifying the virulence factors of FAdV-11 strains. The animal challenge model developed in our study will allow precise evaluation of the efficacy of potential FAdV-11 vaccine candidates.

Impact of Human Adenovirus 36 on Embryonated Chicken Eggs: Insights into Growth Mechanisms.

Human adenovirus 36 (HAdV-D36) is presently the sole virus identified to be associated with an elevated risk of obesity in both humans and animals. However, its impact on embryonated chicken eggs (ECEs) remains unexplored. This study endeavoured to examine the influence of HAdV-D36 on embryonic development by utilizing embryonated chicken eggs as a dynamic model. To simulate various infection routes, the allantoic cavity and the yolk sac of ECEs were inoculated with HAdV-D36. Subsequently, embryos from both the experimental (inoculated with virus) and control (inoculated with PBS) groups were weighed and subjected to daily histological examination. The daily embryo weights were assessed and compared between groups using the Shapiro-Wilk test. Histopathological changes in tissues were examined and compared between the tested and control groups to ascertain physiological alterations induced by the virus. Our study confirmed a significant increase in the body weight of ECEs. However, this phenomenon was not attributable to adipose tissue development; rather, it was characterized by an augmented number of cells in all observed tissues compared to control subjects. We posit that HAdV-D36 may impact developing organisms through mechanisms other than enhanced adipose tissue development. Specifically, our findings indicate an increased number of cells in all tissues, a phenomenon that occurs through an as-yet-unexplored pathway.

Effects of in ovo injection of bacterial peptides and CpG-ODN on Salmonella enterica serovar Heidelberg infection in specific pathogen-free (SPF) chicks

ABSTRACT The increasing prevalence of pathogenic and antimicrobial-resistant isolates of Salmonella Heidelberg (SH) represents a global concern. Consequently, novel strategies for preventing and controlling infections caused by this bacterium are needed. This study aimed to investigate the protective effects of in ovo injection with a formulation based on bacterial peptides, either alone or in combination with oligodeoxynucleotides containing cytosine-phosphodiester-guanine (CpG-ODN), against SH infection in chicks. Genomic data of SH available in public databases were analyzed to select amino acid sequences of structural proteins or those with greater relevance for intestinal colonization. These sequences were subjected to linear epitope prediction tools to identify highly immunogenic peptides. SPF eggs (n = 180) were incubated and injected via the allantoic cavity at day 18 of incubation. Results showed that in ovo injection of peptides + CpG-ODN reduced SH colonization in the caecal content during the first week post-infection, although it did not reduce overall faecal excretion throughout the study. Furthermore, CpG-ODN injection may positively affect intestinal health, as evidenced by reduced crypt depth at 21 dpi and increased villus height at 28 dpi. Levels of secretory IgA did not differ between chicks in any groups, and no detectable SH counts were found in the livers of chicks in any of the infected groups throughout the study. In conclusion, in ovo injection with peptides + CpG-ODN may help reduce caecal colonization by SH in the early stages of infection, but it does not impact total faecal excretion. Additionally, CpG-ODN injection may improve intestinal health parameters in chicks. RESEARCH HIGHLIGHTS Peptides + CpG-ODN reduced SH in caeca at the first week post-infection. Administered formulations did not reduce SH-faecal excretion. Levels of intestinal IgA were similar between all groups. CpG-ODN improved some parameters associated with chick intestinal health.

Quercetin Alleviates Lipopolysaccharide-Induced Cardiac Inflammation via Inhibiting Autophagy and Programmed Cell Death

ObjectiveThe aim of this study is to explore the potential modulatory role of quercetin against Endotoxin or lipopolysaccharide (LPS) induced septic cardiac dysfunction. MethodsSpecific pathogen-free chicken embryos (n = 120) were allocated untreated control, phosphate buffer solution (PBS) vehicle, PBS with ethanol vehicle, LPS (500 ng/egg), LPS with quercetin treatment (10, 20, or 40 nmol/egg, respectively), Quercetin groups (10, 20, or 40 nmol/egg). Fifteen-day-old embryonated eggs were inoculated with abovementioned solutions via the allantoic cavity. At embryonic day 19, the hearts of the embryos were collected for histopathological examination, RNA extraction, real-time polymerase chain reaction, immunohistochemical investigations, and Western blotting. ResultsThey demonstrated that the heart presented inflammatory responses after LPS induction. The LPS-induced higher mRNA expressions of inflammation-related factors (TLR4, TNFα, MYD88, NF-κB1, IFNγ, IL-1β, IL-8, IL-6, IL-10, p38, MMP3, and MMP9) were blocked by quercetin with three dosages. Quercetin significantly decreased immunopositivity to TLR4 and MMP9 in the treatment group when compared with the LPS group. Quercetin significantly decreased protein expressions of TLR4, IFNγ, MMP3, and MMP9 when compared with the LPS group. Quercetin treatment prevented LPS-induced increase in the mRNA expression of Claudin 1 and ZO-1, and significantly decreased protein expression of claudin 1 when compared with the LPS group. Quercetin significantly downregulated autophagy-related gene expressions (PPARα, SGLT1, APOA4, AMPKα1, AMPKα2, ATG5, ATG7, Beclin-1, and LC3B) and programmed cell death (Fas, Bcl-2, CASP1, CASP12, CASP3, and RIPK1) after LPS induction. Quercetin significantly decreased immunopositivity to APOA4, AMPKα2, and LC3-II/LC3-I in the treatment group when compared with the LPS group. Quercetin significantly decreased protein expressions of AMPKα1, LC3-I, and LC3-II. Quercetin significantly decreased the protein expression to CASP1 and CASP3 by immunohistochemical investigation or Western blotting in treatment group when compared with LPS group. ConclusionQuercetin alleviates cardiac inflammation induced by LPS through modulating autophagy, programmed cell death, and myocardiocytes permeability.

Quercetin Alleviates Inflammation and Energy Deficiency Induced by Lipopolysaccharide in Chicken Embryos.

Energy deficiency causes multiple organ dysfunctions after LPS induction. Quercetin is a phenolic compound found in herbal medicines. However, the effects of quercetin in alleviating LPS-induced energy deficiency remain unclear. In the present study, an in vivo LPS-induced inflammation model was established in chicken embryos. Specific pathogen-free chicken embryos (n = 120) were allocated to control, PBS with or without ethanol, quercetin (10, 20, or 40 nmol, respectively), and LPS (125 ng/egg) with or without quercetin groups. Fifteen day old embryonated eggs were injected with the abovementioned solutions via the allantoic cavity. On embryonic day 19, the tissues of the embryos were collected for histopathological examination using frozen oil red O staining, RNA extraction, real-time quantitative polymerase chain reaction, and immunohistochemical investigations. The glycogen and lipid contents in the liver increased after LPS stimulation as compared with the PBS group, whereas quercetin decreased the accumulation as compared with the LPS group. The mRNA expressions of AMPKα1 and AMPKα2 in the duodena, ceca, and livers were upregulated after LPS induction as compared with the PBS group, while quercetin could downregulate these expressions as compared with the LPS group. The immunopositivity of AMPKα2 in the villus, crypt, lamina propria, tunica muscularis, and myenteric plexus in the duodena and in the cytoplasms of hepatocytes significantly increased after LPS induction when compared with the PBS group (p < 0.01), whereas the immunopositivity to AMPKα2 in the quercetin treatment group significantly decreased when compared with the LPS group (p < 0.01 or p < 0.05). The LPS-induced high expressions of transcription factor PPARα and glucose transporter (SGLT1) were blocked by quercetin in the duodena, ceca, and livers. Quercetin treatment improved the LPS-induced decrease in APOA4 in the duodena, ceca, and livers. The mRNA expression of PEPT1 in the duodena and ceca increased after LPS challenge, whereas quercetin could downregulate PEPT1 gene expression. These data demonstrate that quercetin improved the energy deficiency induced by LPS in chicken embryos. The LPS-induced inflammation model was established to avoid the effect of LPS exposure from the environment and intestinal flora. The results form the basis the administration of quercetin pretreatment (in ovo infection) to improve the energy state of chicken embryos and improve the inflammation response.

Effect of deoxynivalenol on inflammatory injury on the glandular stomach in chick embryos

Deoxynivalenol (DON) has a strong toxic effect on the gastrointestinal mucosa of poultry. In this study, we evaluated chicken embryo development and glandular stomach damage to clarify the immunotoxic effects of DON injected through the allantoic cavity of chicken embryos. The glandular stomach index, routine blood indices, plasma inflammatory factors, pathological changes in the glandular stomach, and transcriptome results were analyzed in the hatching chicks. The results showed that DON was supertoxic to chicken embryos, causing edema, shedding, and bleeding of the mucosa of the glandular stomach, which triggered inflammatory reactions. As the toxin concentration increased, the immune system was successively activated and inhibited, and regulation was carried out by the differential regulation of the mitogen-activated protein kinase (MAPK) signal pathway. These results suggested that the immunotoxic effect of DON on the glandular stomach of chicken embryos was closely related to the regulation of the MAPK signaling pathway.

Research Note: Isolation and complete genome analysis of a genotype II goose astrovirus in Sichuan Province, China

The emergence of Goose astrovirus (GoAstV) has led to the gout in geese. This study aimed to isolate and identify the GoAstV from diseased goslings in Sichuan Province, China, followed by performing whole genome phylogenetic analysis of the isolate. The GoAstV was successfully isolated by inoculating the diseased gosling liver and kidney homogenate into the 11-day-old goose embryo allantoic cavity for 3 passages, and the isolate was named as GoAstV-C2 strain. The virus particles were spherical, without capsule, and the size was about 28 nm under transmission electronic microscope. The complete genome length of GoAstV-C2 was 7.035 nt, and the whole genome sequence phylogenetic analysis revealed that it belongs to the GoAstV genotype II (GoAstV-II) subgenotype IIc. The isolated GoAstV-C2 strain was able to be stably passaged in the goose embryo and uric acid sedimentation was observed. The complete genome bioinformation of GoAstV-C2 determined the evolutionary characteristics of the GoAstV isolated from Sichuan, China. This finding lays a foundation for the development of preventive measures, effective vaccines, and therapeutic drugs.

Quercetin Ameliorates Lipopolysaccharide-Induced Duodenal Inflammation through Modulating Autophagy, Programmed Cell Death and Intestinal Mucosal Barrier Function in Chicken Embryos.

Diarrhea has been a global health problem for centuries, and the treatment has become increasingly difficult duo to the antibiotics overuse and resistance. Quercetin is a common flavonoid of extracts of vegetables, fruits, and traditional Chinese herbs, however, the mechanism of quercetin alleviating LPS-induced duodenal inflammation remains elusive. Specific pathogen-free chicken embryos (n = 120) were allocated to groups including control, PBS with or without alcohol, LPS (125 ng/egg) with or without quercetin (10, 20, or 40 nmol/egg, respectively), and quercetin groups (10, 20, or 40 nmol/egg). Fifteen day-old embryonated eggs were inoculated with abovementioned solutions via the allantoic cavity. At embryonic day 19, the duodena of the embryos were collected for histopathological examination, RNA extraction and real-time polymerase chain reaction, immunohistochemical investigations, and Western blotting. The results demonstrated quercetin enhanced the inflammatory cell infiltration in the Peyer's patch of the intestinal mucosa after LPS induction. The LPS-induced expressions of these inflammation-related factors (TLR4, IL-1β, MMP3, MMP9, NFKB1, IFNγ, IL-8, IL-6) were completely blocked by quercetin. Quercetin also decreased the protein expression of TLR4, IL-1β, MMP3, and MMP9 after LPS induction. Quercetin could down-regulate autophagy gene expression (ATG5, LC3-1, LC3-2, and LKB1), and decreased the protein expression of ATG5, and LC3-1/LC3-2 after LPS induction. Quercetin treatment prevented LPS-induced increases of the gene expressions of programmed cell death factors (TNFα, Fas, CASP1, CASP3, CASP12, Drp1, and RIPK1); meanwhile, quercetin decreased the protein expression of CASP1 and CASP3 after LPS challenge. LPS reduced the gene expression of mucin 2, but upregulated the mRNA and protein expression of claudin 1, occludin, and ZO-1, and this was balanced by quercetin. This evidence suggests that quercetin can alleviate duodenal inflammation induced by LPS through modulating autophagy, programmed cell death, intestinal barrier function.

Assessment of the best inoculation route for virulotyping Enterococcus cecorum strains in a chicken embryo lethality assay

ABSTRACT The study aim was to determine the best inoculation route for virulotyping Enterococcus cecorum in a chicken embryo lethality assay (ELA). Twenty-eight genetically different strains were used. Fourteen strains were isolated from cloaca swabs of broiler reproduction chickens (cloaca strains) and 14 strains from broilers with E. cecorum lesions (lesion strains). In all ELAs, 12-day incubated embryonated broiler eggs were inoculated with approximately 100 colony-forming units of E. cecorum/egg. Twenty embryos per inoculation route and strain were used in each of three experiments. In Experiment 1, four cloaca and four lesion strains were inoculated via various routes, i.e. albumen, amniotic cavity, allantoic cavity, chorioallantoic membrane, intravenous or air chamber. The albumen inoculation route showed low mortality with cloaca strains, high mortality with lesion strains and the largest difference in mortality between these groups of strains (≥60%). This route was therefore used in subsequent experiments. In Experiment 2, the same strains were used to test reproducibility, which proved to be generally good. All 28 strains were thereafter used in Experiment 3. In the three experiments, mortality caused by cloaca and lesion strains ranged from 0-25% and from 15-100%, respectively. Recovery rates, assessed in all experiments after albumen inoculation, were significantly lower from eggs inoculated with cloaca strains, compared to lesion strain-inoculated eggs (P < 0.05). However, the bacterial load of eggs with positive recovery was similar in both groups. In conclusion, the albumen inoculation route appeared to be the best to virulotype E. cecorum strains. RESEARCH HIGHLIGHTS The albumen route is the best to differentiate between E. cecorum strains. Egg albumen likely affects cloaca E. cecorum strains more than lesion strains. Based on SNPs, E. cecorum cloaca strains are clustered as well as lesions strains.

Prevalence and associated risk factors of avian influenza A virus subtypes H5N1 and H9N2 in LBMs of East Java province, Indonesia: a cross-sectional study

Background Avian influenza A virus subtypes H5N1 and H9N2 are contagious zoonotic diseases that are circulating in Indonesia and have raised increasing concern about their potential impacts on poultry and public health. A cross-sectional study was carried out to investigate the prevalence and associated risk factors of avian influenza A virus subtypes H5N1 and H9N2 among poultry in the live bird markets of four cities in East Java province, Indonesia. Methods A total of 600 tracheal and cloacal swabs (267 from backyards, 179 from broilers, and 154 from layers) from healthy birds were collected. The samples were inoculated into specific pathogenic-free embryonated eggs at 9-day-old via the allantoic cavity. qRT-PCR was used for further identification of avian influenza. Results The overall prevalence of circulating influenza A virus subtypes H5N1 and H9N2 was 3.8% (23/600, 95%CI [0.0229–0.0537]). Prevalence was higher in backyards at 5.99% (16/267) followed by broilers (2.23% (4/179)) and layers (1.68% (3/154)). The final multivariable model revealed five risk factors for H9N2 infections: presence of ducks (p = 0.003, OR = 38.2), turkeys (p = 0.017 OR = 0.032), and pheasants in the stall (p = 0.04, OR = 18.422), dry (p = 0.006) and rainy season (p &lt; 0.001), and household birds (p = 0.002) and seven factors for H5N1 infections including: observing rodents (p = 0.036, OR = 0.005), stray dogs access (p = 0.004 OR ≤ 0.001), presence of turkeys (p = 0.03 OR = 0.007), chukars/partridges (p = 0.024 OR = 2500), and peafowls in the stalls (p = 0.0043 OR ≤ 0.001), rainy season (p = 0.001) and birds from the household sources (p = 0.002) in the live bird markets. Conclusions The findings of the current study illustrate the recurring infection and presence of both avian influenza viruses and associated risk factors in the surveyed marketplaces. Effective protective measures and mitigation strategies for risks outlined in this study could help to reduce the burden of H5N1 and H9N2 AI subtypes into the live bird markets of Indonesia.

Electromagnetic Force-Driven Needle-Free in Ovo Injection Device.

Needle-free injections are mainly used for administering human or mammalian vaccines or drugs. However, poultry vaccines, in ovo injections to embryos, subcutaneous injections to chickens, and intramuscular injections are administered using needle injections. This article presents a new needle-free in ovo injection device method that uses push-pull solenoids to eject liquid jets, mainly for embryonic eggs of chickens. Furthermore, our study investigated the suitable jet pressures for using this method and the post-injection hatching rates in 18-day-old embryonic eggs. Using this method, we could deliver the liquid to the allantoic and amniotic cavities or the muscle tissue through the egg membrane of the air chamber using a jet pressure of ~6–7 MPa or ~8 MPa. After injecting 0.25 mL of 0.9% saline into 18-day-old Lohmann breed layer embryonic eggs and specific pathogen-free (SPF) embryonic eggs at a jet pressure of ~7 MPa, we observed hatching rates of 98.3% and 85.7%, respectively. This study’s electromagnetic needle-free in ovo injection device can apply vaccine or nutrient solution injection for embryo eggs and serve as a reference for future studies on needle-free in ovo injection automation systems, jet pressure control, and injection pretreatment processes.

Pathogenesis of Velogenic Genotype VII.1.1 Newcastle Disease Virus Isolated from Chicken in Egypt via Different Inoculation Routes: Molecular, Histopathological, and Immunohistochemical Study.

Simple SummaryNewcastle disease (ND) is a worldwide distributed highly infectious disease of wild and domestic birds. Regardless of vaccination procedures, little information is available about the pathogenicity and genetic characteristics of the circulating virus in the Egyptian environment. The current study was undertaken to estimate the role of the different inoculation routes in the pathogenicity of NDV. Therefore, induction of the infection was done with NDV-CH-EGYPT-F42-DAKAHLIA-2019 (VNDV) isolated strain in 28 day old white Ross male broiler chicks via intraocular, choanal, intranasal, mixed oculo-nasal routes. Interestingly, a series of findings including mortality rates, severity of clinical and histopathological lesions, and intensity of viral nucleoprotein immunolabeling were reported to be different according to the routes of inoculation. Clearly, the present findings provide novel descriptive and comparative histopathological and immunohistochemical findings about this virus in Egypt, and the obtained data may be useful for different vaccination strategies against NDV. Newcastle disease virus (NDV) remains a constant threat to the poultry industry. There is scarce information concerning the pathogenicity and genetic characteristics of the circulating velogenic Newcastle disease virus (NDV) in Egypt. In the present work, NDV was screened from tracheal swabs collected from several broiler chicken farms (N = 12) in Dakahlia Governorate, Egypt. Real-time reverse transcriptase polymerase chain reaction (RRT-PCR) was used for screening of velogenic and mesogenic NDV strains through targeting F gene fragment amplification, followed by sequencing of the resulting PCR products. The identified strain, namely, NDV-CH-EGYPT-F42-DAKAHLIA-2019, was isolated and titrated in the allantoic cavity of 10 day old specific pathogen-free (SPF) embryonated chicken eggs (ECEs), and then their virulence was determined by mean death time (MDT) and intracerebral pathogenicity index (ICPI). The pathogenicity of the identified velogenic NDV strain was also assessed in 28 day old chickens using different inoculation routes as follows: intraocular, choanal slit, intranasal routes, and a combination of both intranasal and intraocular routes. In addition, sera were collected 5 and 10 days post inoculation (pi) for the detection of NDV antibodies by hemagglutination inhibition test (HI), and tissue samples from different organs were collected for histopathological and immunohistochemical examination. A series of different clinical signs and postmortem lesions were recorded with the various routes. Interestingly, histopathology and immunohistochemistry for NDV nucleoprotein displayed widespread systemic distribution. The intensity of viral nucleoprotein immunolabeling was detected within different cells including the epithelial and endothelium lining, as well as macrophages. The onset, distribution, and severity of the observed lesions were remarkably different between various inoculation routes. Collectively, a time-course comparative pathogenesis study of NDV infection demonstrated the role of different routes in the pathogenicity of NDV. The intranasal challenge was associated with a prominent increase in NDV lesions, whereas the choanal slit route was the route least accompanied by severe NDV pathological findings. Clearly, the present findings might be helpful for implementation of proper vaccination strategies against NDV.

Avian paramyxovirus 4 isolated from the mallard (&lt;i&gt;Anas platyrhynchos&lt;/i&gt;, Linnaeus, 1758): the first case detected in the Western Caspian region

Aim . The work is aimed at exploring the molecular and genetic properties of avian paramyxovirus 4 (APMV‐4) isolated from the mallard duck. Material and Methods . In 2018, as a part of an influenza A virus surveillance programme, biological samples were collected from wild birds on Lake Adzhi (Papas) in the Republic of Dagestan. The isolation of virus strains from biological samples was performed using 10‐day‐old special pathogen‐free chicken eggs. The primary detection of hemagglutinating agents in the fluid of the allantoic cavity was carried out via hemagglutination assay. The presence of RNA of the influenza A virus was determined using Real Time PCR. Then, for samples negative in PCR for the influenza A virus, the complete genome DNA sequences were established using NGS sequencing. It was determined that the APMV‐4 strain obtained belonged to intraspecific genetic lines was determined using phylogenetic analysis. Results . APMV‐4 strain was isolated for the first time in the Western Caspian region. Phylogenetic analysis of the complete genome and gene F showed that the APMV‐4/mallard/Dagestan/92d/2018 strain belongs to the Eurasian clade of subgenotypes. Analysis of amino acid substitutions showed that the cleavage site of the fusion protein is monobasic which is characteristic of lentogenic strains. Conclusion . During the surveillance program for influenza A viruses in autumn 2018, the APMV‐4 strain was isolated from the mallard duck. Analysis revealed that the APMV‐4 strain was closely phylogenetically related to strains isolated from two wild and one domestic duck from China. This suggests an interconnection between the bird populations in China and the Western Caspian region within the West Asian‐East African migration route.

Sensitivity of infl uenza virus strains isolated from various regions of Kazakhstan in 2018–2019 to antiviral drugs

The purpose of the study was to examine susceptibility of the Kazakhstan strains of infl uenza A/H1N1 and type B viruses, isolated from various regions of Kazakhstan in 2018–2019, to antiviral drugs. Materials and methods. The susceptibility analysis of 20 strains of infl uenza A/H1N1 and B viruses was carried out with chemotherapeutic agents including Remantadine, Tamifl u, Arbidol, and Ingavirin. Viruses were cultured in the allantoic cavity of developing 10-day-old chicken embryos for 48 hours at 36 °C. The hemagglutinating activity was determined according to the conventional method on 96-well plates using 0.75% chicken red blood cell suspension; the infectivity was calculated by the Reed-Muench method. The sensitivity of virus strains to diff erent concentrations of antiviral drugs was evaluated by the level of reproductive suppression of 100 lg EID50/0.2 ml of virus in chicken embryos. Statistical analysis was performed with the use of Microsoft Offi ce Excel 2010 software. Results. A study of sensitivity to chemotherapeutic agents demonstrated heterogeneity of Kazakhstan 2018–2019 infl uenza A and B viruses population on this feature. The sensitivity to Tamifl u was found in all Kazakhstan strains of infl uenza A/H1N1 virus and three type B strains (inhibitory concentration was 0.44–25.38 μg/mL). The reproduction of most viruses was eff ectively inhibited by tamifl u at a concentration of 0.68–3.23 μg/mL. The inhibitory concentration for the three strains of A/H1N1 virus was 7.23–25.38 μg/mL. Remantadin inhibited the reproduction of viruses at higher doses (12.60–25.55 μg/mL). All viruses under study were resistant to Arbidol and Ingavirin. One type B infl uenza virus was found to be weakly sensitive to Ingavirin. Conclusion. The heterogeneity of the infl uenza virus population in their sensitivity to antiviral drugs indicates the need for constant epidemiological surveillance in order to identify drug-resistant variants.

In vivo typing of Escherichia coli obtained from laying chickens with the E. coli peritonitis syndrome

ABSTRACT Fourteen genetically different chicken Escherichia coli strains were biotyped in hens to examine if any E. coli strain at high dose can induce the E. coli peritonitis syndrome (EPS). Moreover, biotyping was performed in embryos and the median lethal dose (LD50) of three strains was determined in hens. Nine strains were obtained from femur marrow and one strain from caecum of hens that had died from EPS. One strain originated from the inflamed pericardium of a broiler and three strains from the cloaca of specified pathogen-free (SPF) broiler breeders. Strains were inoculated intratracheally into separate groups of 32 productive SPF White Leghorn (WL) hens at a dose of 7.8–9.2 log10 colony forming units (CFU) per hen and into the allantoic cavity of separate groups of 20 SPF WL embryos incubated during 14 days in a dose of 4.2–4.6 log10 CFU per embryo. The embryo test was replicated. Bone marrow and pericardium strains induced EPS, the other strains did not. Based on mortality in hens, EPS-inducing strains could be classified as very virulent (59–100% mortality), moderately virulent (38% mortality) and low virulent (6% mortality). In productive SPF WL hens, the LD50 of three very virulent strains ranged from <2.7 to 5.3 log10 CFU. Virulent and avirulent strains killed 60–95% and 0–30% of embryos, respectively. The embryo lethality test, which showed good reproducibility, did not discriminate within virulent strains, but can nevertheless be considered as a useful alternative for biotyping E. coli in productive hens. RESEARCH HIGHLIGHTS Even at high doses, no E. coli strain could induce EPS. Substantial differences in virulence exist within very virulent E. coli strains. The embryo lethality test is a useful alternative for biotyping E. coli in laying hens. Broiler colibacillosis may represent a source of EPS strains for layers and vice versa.

2018–2019 antiviral drug sensitivity of the influenza virus strains isolated from various regions of Kazakhstan

Influenza is a serious public health problem. The ability of influenza virus to change upon replication is the most serious issue for practical medicine and virology, which can fundamentally alter virus biological properties, such as infectivity and virulence. The high mutational variability of influenza viruses can contribute to rapidly emerging drug resistance. Therefore, the study of antiviral drug sensitivity among influenza viruses is necessary to justify proper drug use for treatment and prevention of influenza infection. The aim of the study was to examine antiviral drug susceptibility of influenza A/H1N1 and B virus strains isolated from various regions of Kazakhstan in the years 2018–2019. Materials and methods. The susceptibility analysis of 20 strains of influenza A/H1N1 and B viruses was carried out by using chemotherapeutic agents including Remantadine, Tamiflu, Arbidol, and Ingavirin. Viruses were cultured in the allantoic cavity of developing 10-day-old chicken embryos for 48 hours at 36оC. The hemagglutinating activity was determined according to the standard method on 96-well plates using 0.75% chicken red blood cell suspension; the infectivity was calculated by the Reed–Muench method. The susceptibility of virus strains to different concentrations of antiviral drugs was evaluated by the level of virus reproductive suppression of 100 lg EID50/0.2 ml in chicken embryos. Statistical analysis was performed using Microsoft Office Excel 2010 software. Results. A study of susceptibility to chemotherapeutic agents demonstrated heterogeneity of influenza A and B virus population isolated in Kazakhstan during the 2018–2019 period. The susceptibility to tamiflu was found in all Kazakhstan strains of influenza A/H1N1 virus and three type B strains (inhibitory concentration was 0.44–25.38 μg/mL). The reproduction of most viruses was effectively inhibited by Tamiflu at a concentration of 0.68–3.23 μg/mL. The inhibitory concentration for three strains of A/H1N1 virus was 7.23–25.38 μg/mL. Remantadine inhibited reproduction of viruses at higher doses (12.60–25.55 μg /mL). All investigated viruses were resistant to Arbidol and Ingavirin. A single type B influenza virus strain was found to be weakly susceptible to Ingavirin. Conclusion. The heterogeneity of influenza virus population in susceptibility to antiviral drugs suggest a need for constant epidemiological surveillance in order to identify drug-resistant variants.

Isolation and Molecular Characterization of Newcastle Disease Virus in Layers

Background: Newcastle disease (ND) in spite of the availability of vaccines remains a constant threat to poultry producers worldwide. It is prevalent in Indian subcontinent and leads to economic losses. The present study was aimed with isolate and identify virulent Newcastle disease virus (NDV) in layer poultry from field outbreaks.Methods: Total 47 samples consisting of nasal (05), oropharyngeal (13) and cloacal swabs (11) and tissue samples consisting of trachea (07), lungs (06), larynx (05) were collected from layer birds. For isolation of NDV swab and tissue samples were inoculated in 9-11 days old embryonated eggs via allantoic cavity route. After preparing the viral inoculum, 47 suspected samples (29 swab and 18 tissue samples) were inoculated in 141 embryonated eggs to isolate the virus.Result: Out of 47 samples 10 (21.27%) samples were positive for HA activity. All the 10 isolates showing HA activity subjected to Reverse-Transcriptase PCR of F gene and 6 were found positive in RT-PCR for F1 gene. The PCR amplified product showed amplicon at 356 bp and 254 bp positive for F1 and F2 gene, respectively. On basis of F gene, 06 (50%) isolates were considered as virulent Newcastle Disease Virus. One isolate sequence was submitted at NCBI with accession MT890653 On phylogenetic analysis MT890653 designated as Class II/ genotype II/ virulent strain and had the motif 112R-R-R-K-R-F117 at the cleavage site of the fusion protein.

Recent detection and Genotypic characterization of duck hepatitis virus A type 1and H5 avian influenza virus from ducks.

Objective: To identify some viruses affecting ducks by Serological and molecular studies.Design: Diagnostic study.Procedures: Twenty pooled liver samples were collected from ducklings less than 2 weeks showed nervous signs and high mortality suspected to be infected by duck hepatitis virus (DHV). All samples were inoculated in allantoic cavity of SPF embryonated chicken eggs (ECGs) for three successive passages. Isolates were serologically identified by agar gel precipitation (AGPT) and fluorescent antibody technique (FAT) by using DHAV-1 antibodies as well. Also, other 20 pooled lung and pancreas samples from ducks more than one month suspected to be infected by avian influenza virus (AIV) showed nervous signs; ataxia and torticolus. Suspected AIV samples were tested by reverse transcriptase polymerase chain reaction (RT-PCR) of H5 gene. Finally, sequencing and sequencing analysis of our RT-PCR products of DHAV-1 3D gene and AIV H5 gene with that published in GenBank.Results: Twelve out of 20 local isolates of DHAV-1 were positive in AGPT and 15 gave positive result in FAT. Twelve samples suspected to be AIV gave positive HA reaction. By RT-PCR 5 selected isolates of DHAV-1 and 5 out of 10 tested samples of AIV were positive and gave the expected bands. Our DHAV-1 isolates showed 100% identity to other Egyptian sequences from GenBank and H5 AIV showed 96-97% identity.Conclusion and clinical relevance: Both DHAV-1 and H5 AIV still in ducks could be identified by serological and molecular methods.