Antibiofilm activity of a lytic Salmonella phage on different Salmonella enterica serovars isolated from broiler farms.

Bacteriophages have been mainly used in treating infections caused by planktonic bacterial cells in the veterinary sector. However, their applications as antibiofilm agents have received little attention. Accordingly, a previously isolated Salmonella infecting Siphoviridae phage was investigated for host range against 15 Salmonella enterica isolates (S. Cape, S. Gallinarum, 4 S. Enteritidis, 3 S. Montevideo, S. Uno, S. Oritamerin, S. Belgdam, S. Agona, S. Daula, and S. Aba) recovered from the litters of commercial broiler farms. All S. enterica isolates were examined for their biofilm activity using a microtiter plate assay and for adrA, csgD, and gcpA genes using conventional PCR. The phage efficacy against established biofilms produced by the selected seven S. enterica isolates (S. Gallinarum, S. Enteritidis, S. Montevideo, S. Uno, S. Oritamerin, S. Belgdam, and S. Agona) was assessed using microtiter plate assay and reverse transcriptase real-time PCR over different incubation times of 5 and 24 h. All S. enterica isolates were strong biofilm formers. Moreover, the phage effectively reduced the biofilm activity of the established S. enterica biofilms in the microtiter plate assay using the independent sample t-test (P < 0.050). Furthermore, the relative expression levels of csgD, gcpA, and adrA genes in the biofilm cells of S. enterica isolate after phage treatment were significantly up-regulated to variable degrees using the independent sample t-test (P < 0.050). In conclusion, the present study revealed the potential use of Salmonella phage in reducing established biofilms produced by S. enterica serovars isolated from broiler farms.

Initiation and regulation of immune responses to immunization with whole inactivated vaccines prepared from two genetically and antigenically distinct lineages of Egyptian influenza A virus subtype H5N1.

Following the introduction of highly pathogenic avian influenza (HPAI) virus subtype H5N1, the Egyptian government implemented a massive poultry vaccination campaign as the cornerstone of its policies to control the virus. The efficacy of vaccination has been evaluated primarily by measuring titers of antibodies inhibiting the hemagglutinating activity of the viral hemagglutinin (HA). However, other aspects of the host response remain poorly understood. In the present study, in addition to hemagglutination inhibition (HI) titers, cytokine profiles were examined and IFNγ concentrations were measured in vivo after immunization with a whole inactivated virus (WIV) prepared from a classical strain of clade 2.2.1.2 (C121) and an antigenic drift variant of clade 2.2.1.1 (V1063). The results revealed an earlier response and higher HI titers and IFNγ levels in sera from chickens immunized with C121, accompanied by significantly higher expression of IL8, IL10, and IL18 in the spleen and IL6 and IL10 in the bursa, compared to those immunized with V1063. Furthermore, stimulation of the HD11 cell line with C121 induced gradual upregulation of pro-inflammatory cytokines, which was observed at 24 hours post-inoculation (hpi), and became more pronounced at 48 and 72 hpi, accompanied by upregulation of IFNα. Conversely, V1063 induced very early transient higher expression of pro-inflammatory cytokines at 3 and 6 hpi accompanied by upregulation of IL10, which then decreased at 24, 48 and 72 hpi. In summary, our results provide evidence of a correlation between adaptive immune responses induced by WIVs and higher expression of IL10 and IL18 in addition to early induction of IFNα. These findings could be used to improve immune responses induced by WIVs.

Surveillance on A/H5N1 virus in domestic poultry and wild birds in Egypt.

BACKGROUND: The endemic H5N1 high pathogenicity avian influenza virus (A/H5N1) in poultry in Egypt continues to cause heavy losses in poultry and poses a significant threat to human health. METHODS: Here we describe results of A/H5N1 surveillance in domestic poultry in 2009 and wild birds in 2009-2010. Tracheal and cloacal swabs were collected from domestic poultry from 22024 commercial farms, 1435 backyards and 944 live bird markets (LBMs) as well as from 1297 wild birds representing 28 different types of migratory birds. Viral RNA was extracted from a mix of tracheal and cloacal swabs media. Matrix gene of avian influenza type A virus was detected using specific real-time reverse-transcription polymerase chain reaction (RT-qPCR) and positive samples were tested by RT-qPCR for simultaneous detection of the H5 and N1 genes. RESULTS: In this surveillance, A/H5N1 was detected from 0.1% (n = 23/) of examined commercial poultry farms, 10.5% (n = 151) of backyard birds and 11.4% (n = 108) of LBMs but no wild bird tested positive for A/H5N1. The virus was detected from domestic poultry year-round with higher incidence in the warmer months of summer and spring particularly in backyard birds. Outbreaks were recorded mostly in Lower Egypt where 95.7% (n = 22), 68.9% (n = 104) and 52.8% (n = 57) of positive commercial farms, backyards and LBMs were detected, respectively. Higher prevalence (56%, n = 85) was reported in backyards that had mixed chickens and waterfowl together in the same vicinity and LBMs that had waterfowl (76%, n = 82). CONCLUSION: Our findings indicated broad circulation of the endemic A/H5N1 among poultry in 2009 in Egypt. In addition, the epidemiology of A/H5N1 has changed over time with outbreaks occurring in the warmer months of the year. Backyard waterfowl may play a role as a reservoir and/or source of A/H5N1 particularly in LBMs. The virus has been established in poultry in the Nile Delta where major metropolitan areas, dense human population and poultry stocks are concentrated. Continuous surveillance, tracing the source of live birds in the markets and integration of multifaceted strategies and global collaboration are needed to control the spread of the virus in Egypt.

Effect of cocirculation of highly pathogenic avian influenza H5N1 subtype with low pathogenic H9N2 subtype on the spread of infections.

Widespread prevalence of avian influenza H9N2 subtype in the Middle East region and its detection in Egypt in quail in early summer 2011 added another risk factor to the Egyptian poultry industry in addition to highly pathogenic H5N1 subtype. This situation increases the need for further surveillance and investigation of H9N2 viruses in commercial and household chickens. This work describes detection and genetic characterization of recently isolated H9N2 viruses from chicken flocks. Parallel detection and genetic characterization of H5N1 viruses from infections in poultry has also been done to compare the prevalence of the two subtypes in close geographic locations in Egypt. Phylogenetic analysis of the HA gene showed that the Egyptian isolates of H9N2 were grouped together within the quail/Hong Kong/G1/97-like lineage, similar to the circulating viruses in the Middle East, with very close phylogeny to the Israeli viruses. The prevalence of H5N1 viruses from cases recorded in poultry in the nearby areas revealed a marked decrease in disease incidence in commercial broilers but an increased incidence in household birds. The genetic characterization of the H5N1 viruses indicated predominance of the classic 2.2.1 subclade, with evolution of new viruses and no detection for the variant 2.2.1.1 subclade. The cocirculation of the two subtypes, H5N1 and H9N2, of avian influenza may affect the limit of spread and the epizootiologic pattern of the infections for both subtypes, especially when different vaccination and biosecurity approaches are applied in the field level.

Bacteriological and molecular study of Salmonella species associated with central nervous system manifestation in chicken flocks.

BACKGROUND AND AIM: Salmonella species often cause systemic health problems in poultry flocks, sometimes including nervous systems manifestations. This impact of Salmonella has rarely been studied. This study aimed to define an alternative pathogenic pathway for Salmonella spp. invasion of brain tissue in chicken flocks. Brain infection produces neurological manifestations; Salmonella strains isolated from brain tissue showed the presences of two virulence genes. Confirmation of the pathway of isolates from intestinal mucosa through the blood-brain barrier was attained using experimental infections in specific pathogen-free (SPF)-day-old chicks through two routes of inoculation. MATERIALS AND METHODS: Isolation of Salmonella spp. from five chicken flocks that showed signs of the central nervous system (CNS) effects were isolated. Isolates were characterized by serotyping, and antimicrobial assays. In addition, virulence profiles were described using detection of virulence plasmid spvC, and Salmonella plasmid sopB. A pathogenicity study of isolates in specific pathogen-free (SPF)-day-old chicks through oral and intracerebral administration performed, and experimental infection in SPF embryonated chicken eggs through intra-yolk and intra-allantoic administration was investigated. Supporting histopathology and immunohistopathology against Salmonella antigen in brain tissue were performed for flock and experimental infections. RESULTS: Three serotypes of Salmonella were isolated from the brains of five flocks (two Salmonella Virchow, two Salmonella Kentucky, and one Salmonella Enteritidis isolates). Phage related gene sopB and plasmid-mediated operon spvC were identified in all isolated strains. The Salmonella strains were re-isolated and identified from the brain and internal organs of post-experimental infected chicks. Infected chicks showed nervous manifestations associated with Salmonella infection. The presence of positively stained Salmonella antigen in brain tissues indicates penetration of the blood-brain barrier by the Salmonella species. CONCLUSION: Our results indicate that some virulent systemic strains of Salmonella spp. can induce CNS manifestations in chicken hosts.

Detection of Novel Goose Parvovirus Disease Associated with Short Beak and Dwarfism Syndrome in Commercial Ducks.

Derzsy's disease causes disastrous losses in domestic waterfowl farms. A genetically variant strain of Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) was named novel goose parvovirus (NGPV), which causes characteristic syndrome in young ducklings. The syndrome was clinically characterized by deformity in beaks and retarded growth, called short beaks and dwarfism syndrome (SBDS). Ten mule and pekin duck farms were investigated for parvovirus in three Egyptian provinces. Despite low recorded mortality rate (20%), morbidity rate was high (70%), but the economic losses were remarkable as a result of retarded growth and low performance. Isolation of NGPV was successful on primary cell culture of embryonated duck liver cells with a clear cytopathic effect. Partial gene sequence of the VP1 gene showed high amino acids identity among isolated strains and close identity with Chinese strains of NGPV, and low identity with classic GPV and MDPV strains. To the best of our knowledge, this can be considered the first record of NGPV infections in Egypt.

Efficacy of Live Attenuated Vaccine and Commercially Available Lectin Against Avian Pathogenic E. coli Infection in Broiler Chickens.

In this study, the protective efficacy of an E. coli live attenuated vaccine was compared to the preventive administration of lectin preparation before the challenge. Two hundred broiler chicks were divided into eight equal groups. The first group was used as a negative control group. Three groups were vaccinated at day 1 with the avian colibacillosis live vaccine of which one group served as a vaccinated nonchallenged group. Another two groups were treated with lectin product (0.5 mL/L drinking water) for three days before the challenge. The last two groups served as challenge control for either E. coli O78 or O125 strains. The challenge was conducted at three weeks of age with either homologous O78 or heterologous O125 E. coli strains, using 0.5 mL/bird of each avian pathogenic E. coli (APEC) strain (~108 colony forming units "CFU"/mL)/subcutaneously. The bodyweight and feed conversion ratios (FCR) were calculated for four weeks. Clinical signs and gross and histopathological lesions were scored at two and seven days post inoculation (dpi). The heart and liver of euthanized chickens at 2 dpi were removed aseptically and homogenized to evaluate pathogenic E. coli colonization. Results showed that live avian colibacillosis vaccine reduced mortalities and APEC colonization in the homologous challenge group but not in the heterologous challenge group. Lectin-treated groups showed 20% and 16% mortality after challenge with E. coli O78 and O125, respectively, and both groups showed performance parameters, clinical signs, and histopathological lesion scores comparable to the negative control group, with variable E. coli colonization of heart and liver. The study demonstrated the efficacy of live attenuated avian colibacillosis vaccine against homologous but not heterologous APEC challenge in broiler chickens. The lectin-containing products can be used as a preventive medication to reduce the clinical impacts of colibacillosis regardless of the challenge strain. Standardization of the evaluation parameters for APEC vaccines is recommended.

The occurrence of disinfectant and antibiotic-resistant genes in Escherichia coli isolated from chickens in Egypt.

AIM: This work aimed to determine the occurrence of antibiotic and disinfectant resistance genes in Escherichia coli isolated from chickens in Egypt. MATERIALS AND METHODS: Organs (liver, lung, heart, yolk sac, and bone marrow) of 1500 chicken samples were collected from diseased chickens suffered from colibacillosis with PM findings as CRD, diarrhea and omphalitis from different governorates of Egypt as: Giza, EL-Bahira, Fayoum, El-Dakahlia, El-Ismalia, and El-Sharkia during 2015-2016. These samples were labeled and transported immediately on ice to the Reference laboratory for quality control on poultry production (RLQP). The samples were cultured onto MacConkey agar and Eosin Methylene Blue Agar. Isolation and identification of the E. coli were performed based on morphology, cultural, staining, and biochemical properties. Antimicrobial resistance test was carried out using disk diffusion method. The PCR employing tetA, qacED1 and qacA/B were carried out for detection of these genes in isolated E.coli. RESULTS: The prevalence of E. coli in chicken was 34%. Predominant serotypes of E. coli which serologically identified were O128, O111, O44, O158, and O2. Antibiotic susceptibility test of E. coli revealed that 100% of isolates were resistant to ampicillin, erythromycin, and sulfamethoxazole-trimethoprim, while 73.53% and 38.23% of them were sensitive for colistin sulfate and levofloxacin, respectively. Antibiotic resistance genes as tetA gene were tested for isolated E. coli and detected by incidence rate of 91.18%. qac resistance genes resembling as qacED1 and qacA/B genes were detected in isolated E. coli 70.6% and 14.7%, respectively. CONCLUSION: E. coli isolated from chickens in Egypt was carried qac and antibiotic-resistant genes that affect the poultry industry.

The occurrence of the multidrug resistance (MDR) and the prevalence of virulence genes and QACs resistance genes in E. coli isolated from environmental and avian sources.

Colibacillosis is a major disease affecting poultry leads to high morbidity and mortality which causing tremendous economic losses worldwide. These economic disparities are amplified among low and middle-income where sanitation and hygiene are challenged by the increasing demand for quality sources of animal protein. With a view to investigating the prevalence of virulence genes and QACs resistance genes as well as monitoring the antibiogram of E. coli strains, a total of 368 specimens were collected from diseased broiler chickens (n = 226) and environmental sources (n = 142) at large-scale poultry farms in Ismailia Governorate, Egypt. The bacteriological examination proved that E. coli prevalence was 26.76% and 50.44% in the farm environment and diseased broilers, respectively. In tandem, the isolated E. coli strains were serogrouped, determining the most common serotypes were O78, O1:H7, O91:H21 and O126. Isolates were tested for antimicrobial susceptibility against 12 antibiotics, screened for 4 virulence genes (iss, papC, eaeA, and cfaI), and screened for 3 QACs resistance genes (qacEΔ1, qacA/B, and qacC/D). All the tested strains were positive for iss and papC genes, only 20.3% of the tested strains were positive for eaeA gene, moreover, the examined strains were negative to CFAI gene. Furthermore, all the tested strains were positive for qacEΔ1, qacA/B, and qacC/D genes. In conclusion; virulence genes (iss, papC) as well as QACs resistance genes are common in avian Pathogenic E. coli and environmental strains and are mainly associated with multi-drug resistance phenomena.

Antibiotic resistance and polymorphism in the quinolone resistance-determining region of Campylobacter spp. isolated from 1-day-old ducklings.

Thirty-three isolates of Campylobacter coli and three isolates of Campylobacter jejuni were recovered from 150 1-day-old ducklings. All isolates were sensitive to chloramphenicol and amikacin, but resistant to sulfamethoxazole-trimethoprim (SXT) by the disc diffusion method. Most isolates were susceptible to tetracycline and erythromycin, but resistant to ofloxacin and ciprofloxacin. Of the 33 C. coli isolates, nine were positive for the tetracycline resistance gene tet(O), although only two of these were resistant to tetracycline in the disc diffusion test. None of the isolates possessed mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene infrequently linked to FQ-resistance. The finding indicated that ducklings may be a source of antibiotic resistant Campylobacter spp. with potential poultry and public health hazard.

Different counteracting host immune responses to clade 2.2.1.1 and 2.2.1.2 Egyptian H5N1 highly pathogenic avian influenza viruses in naïve and vaccinated chickens.

In Egypt, two distinct lineages of H5N1 highly pathogenic avian influenza (HPAI) viruses, "classic 2.2.1.2" and "variant 2.2.1.1" strains, have evolved. The underlying host immune responses counteracting these viruses in chickens remain not well understood. In the present study, the cytokine responses to a classic strain (C121) and those to a variant strain (V1063) were compared in naïve and vaccinated chickens. In naïve chickens, the C121 replicated more efficiently than the V1063. Both the C121 and the V1063 increased interferon (IFN)-γ and interleukin (IL)-10 gene expression at 48 h post inoculation (hpi) in the lung and spleen but the levels of these cytokines were lower in chickens infected with the C121 than those infected with the V1063. In contrast, in chickens vaccinated with inactivated C121-based vaccine, the C121 replicated less than the V1063. Both challenge with the C121 and that with the V1063 did not increase IFN-γ gene expression at 48 hpi; rather, the C121 increased IL-4 gene expression in the lung accompanied with lower viral titer and higher HI titers. These results suggested that the pathogenicity of HPAI viruses correlated with IFN-γ-producing helper and/or cytotoxic T cell responses in naïve chickens, whereas vaccine efficacy to HPAI viruses correlated with IL-4 producing helper T cell responses in the lung in vaccinated chickens. It implies that IL-4 in the lung, in addition to the traditional serum HI titers, could be used to screen novel vaccine strategies, such as strains, adjuvant, prime/boost protocols, against HPAI in chickens.

Epidemiology and molecular characterisation of duck hepatitis A virus from different duck breeds in Egypt.

Duck hepatitis virus (DHV) is an acute highly contagious disease of ducklings caused by three distinct serotypes of duck hepatitis A virus (DHAV), a member of the RNA family Picornaviridae, where serotype 1 is the most widespread serotype worldwide. To date, little if any is known about the prevalence and genetic characterisation of DHAV outside Asia. The current study describes surveillance on DHV in 46 commercial duck farms in Egypt with a history of high mortality in young ducklings from 3 to 15 day-old from 2012 to 2014. Clinical samples were examined by generic RT-PCR assays followed by partial sequence analysis of the 5'UTR, VP1 and 3D genes of the vaccine strain and 15 field viruses. The overall positive rate was 37% (n=17/46). All duck breeds (Pekin, Muscovy, Mallard and Green Winged) were susceptible to the disease with mortality ranged from 15% to 96.7%. Sequence and phylogenetic analyses indicated that the Egyptian strains cluster in the DHAV serotype 1 with Asian viruses and distinguishable from the vaccine strains. So far, this is the first report on the genetic characterisation of DHAV in Egypt. This study may be useful to better understand the epidemiology and evolution of DHAV.