The Microbiota Contributes to the Control of Highly Pathogenic H5N9 Influenza Virus Replication in Ducks.

Ducks usually show little or no clinical signs following highly pathogenic avian influenza virus infection. In order to analyze whether the microbiota could contribute to the control of influenza virus replication in ducks, we used a broad-spectrum oral antibiotic treatment to deplete the microbiota before infection with a highly pathogenic H5N9 avian influenza virus. Antibiotic-treated ducks and nontreated control ducks did not show any clinical signs following H5N9 virus infection. We did not detect any significant difference in virus titers neither in the respiratory tract nor in the brain nor spleen. However, we found that antibiotic-treated H5N9 virus-infected ducks had significantly increased intestinal virus excretion at days 3 and 5 postinfection. This was associated with a significantly decreased antiviral immune response in the intestine of antibiotic-treated ducks. Our findings highlight the importance of an intact microbiota for an efficient control of avian influenza virus replication in ducks.IMPORTANCE Ducks are frequently infected with avian influenza viruses belonging to multiple subtypes. They represent an important reservoir species of avian influenza viruses, which can occasionally be transmitted to other bird species or mammals, including humans. Ducks thus have a central role in the epidemiology of influenza virus infection. Importantly, ducks usually show little or no clinical signs even following infection with a highly pathogenic avian influenza virus. We provide evidence that the microbiota contributes to the control of influenza virus replication in ducks by modulating the antiviral immune response. Ducks are able to control influenza virus replication more efficiently when they have an intact intestinal microbiota. Therefore, maintaining a healthy microbiota by limiting perturbations to its composition should contribute to the prevention of avian influenza virus spread from the duck reservoir.

The Cloacal Microbiome of Five Wild Duck Species Varies by Species and Influenza A Virus Infection Status.

Waterfowl, especially ducks of the genus Anas, are natural reservoir species for influenza A virus (IAV). Duck populations contain nearly all the known diversity of IAVs, and the birds are asymptomatic to infection. Previous work established that IAV infection status is correlated with changes in the cloacal microbiome in juvenile mallards. Here, we analyze five Anas species to determine whether these duck species have similar IAV+ and IAV- cloacal microbiomes, or if the relationships among a host, influenza virus, and the microbiome are species specific. We assessed taxonomic composition of the microbiome, alpha diversity, and beta diversity and found very few patterns related to microbiome and infection status across species, while detecting strong differences within species. A host species-specific signal was stronger in IAV- ducks than IAV+ ducks, and the effect size of host species on the microbiome was three times higher in IAV- birds than IAV+ birds. The mallards and the northern shovelers, the species with highest sample sizes but also with differing feeding ecology, showed especially contrasting patterns in microbiome composition, alpha diversity, and beta diversity. Our results indicate that the microbiome may have a unique relationship with influenza virus infection at the species level.IMPORTANCE Waterfowl are natural reservoir species for influenza A virus (IAV). Thus, they maintain high levels of pathogen diversity, are asymptomatic to the infection, and also contribute to the risk of a global influenza pandemic. An individual's microbiome is a critical part in how a vertebrate manages pathogens and illness. Here, we describe the cloacal microbiome of 300 wild ducks, from five species (four with previously undescribed microbiomes), including both IAV-negative and IAV-positive individuals. We demonstrate that there is not one consistent "flu-like" microbiome or response to flu across species. Individual duck species appear to have unique relationships between their microbiomes and IAV, and IAV-negative birds have a stronger tie to host species than the IAV-positive birds. In a broad context, understanding the role of the microbiome in IAV reservoir species may have future implications for avian disease management.

Gut microbiota-mediated protection against influenza virus subtype H9N2 in chickens is associated with modulation of the innate responses.

Commensal gut microbiota plays an important role in health and disease. The current study was designed to assess the role of gut microbiota of chickens in the initiation of antiviral responses against avian influenza virus. Day-old layer chickens received a cocktail of antibiotics for 12 (ABX-D12) or 16 (ABX-D16) days to deplete their gut microbiota, followed by treatment of chickens from ABX-12 with five Lactobacillus species combination (PROB), fecal microbial transplant suspension (FMT) or sham treatment daily for four days. At day 17 of age, chickens were challenged with H9N2 virus. Cloacal virus shedding, and interferon (IFN)-α, IFN-ß and interleukin (IL)-22 expression in the trachea, lung, ileum and cecal tonsils was assessed. Higher virus shedding, and compromised type I IFNs and IL-22 expression was observed in ABX-D16 chickens compared to control, while PROB and FMT showed reduced virus shedding and restored IL-22 expression to levels comparable with undepleted chickens. In conclusion, commensal gut microbiota of chickens can modulate innate responses to influenza virus subtype H9N2 infection in chickens, and modulating the composition of the microbiome using probiotics- and/or FMT-based interventions might serve to promote a healthy community that confers protection against influenza virus infection in chickens.

Avian Influenza Virus Subtype H9N2 Affects Intestinal Microbiota, Barrier Structure Injury, and Inflammatory Intestinal Disease in the Chicken Ileum.

Avian influenza virus subtype H9N2 (H9N2 AIV) has caused significant losses to the poultry industry due to the high mortality associated with secondary infections attributable to E. coli. This study tries to address the underlying secondary mechanisms after H9N2 AIV infection. Initially, nine day-old specific pathogen-free chickens were assigned to control (uninfected) and H9N2-infected groups, respectively. Using Illumina sequencing, histological examination, and quantitative real-time PCR, it was found that H9N2 AIV caused intestinal microbiota disorder, injury, and inflammatory damage to the intestinal mucosa. Notably, the genera Escherichia, especially E. coli, significantly increased (p < 0.01) at five days post-infection (dpi), while Lactobacillus, Enterococcus, and other probiotic organisms were significantly reduced (p < 0.01). Simultaneously, the mRNA expression of tight junction proteins (ZO-1, claudin 3, and occludin), TFF2, and Muc2 were significantly reduced (p < 0.01), indicating the destruction of the intestinal epithelial cell tight junctions and the damage of mucin layer construction. Moreover, the mRNA expression of proinflammatory cytokines IFN-γ, IL-22, IFN-α, and IL-17A in intestinal epithelial cells were significantly upregulated, resulting in the inflammatory response and intestinal injury. Our findings may provide a theoretical basis for observed gastroenteritis-like symptoms such as diarrhea and secondary E. coli infection following H9N2 AIV infection.

Influenza A virus subtype H9N2 infection disrupts the composition of intestinal microbiota of chickens.

The impact of low pathogenic influenza viruses such as subtype H9N2, which infect the respiratory and the gastrointestinal tracts of chickens, on microbial composition are not known. Twenty-day-old specific pathogen-free chickens were assigned to two treatment groups, control (uninfected) and H9N2-infected (challenged via the oral-nasal route). Fecal genomic DNA was extracted, and the V3-V4 regions of the 16S rRNA gene were sequenced using the Illumina Miseq® platform. Sequences were curated using Mothur as described in the MiSeq SOP. Infection of chickens with H9N2 resulted in an increase in phylum Proteobacteria, and differential enrichment with the genera Vampirovibrio, Pseudoflavonifractor, Ruminococcus, Clostridium cluster XIVb and Isobaculum while control chickens were differentially enriched with genera Novosphingobium, Sphingomonas, Bradyrhizobium and Bifidobacterium. Analysis of pre- and post-H9N2 infection of the same chickens showed that, before infection, the fecal microbiota was characterized by Lachnospiracea and Ruminococcaceae family and the genera Clostridium sensu stricto, Roseburia and Lachnospiraceae incertae sedis. However, post-H9N2 infection, class Deltaproteobacteria, orders Clostridiales and Bacteroidiales and the genus Alistipes were differentially enriched. Findings from the current study show that influenza virus infection in chickens results in the shift of the gut microbiota, and the disruption of the host-microbial homeostasis in the gut might be one of the mechanisms by which influenza virus infection is established in chickens.

Respiratory phagocytes are implicated in enhanced colibacillosis in chickens co-infected with influenza virus H9N2 and Escherichia coli.

1. The aim of this study was to determine the most likely time interval after infection with influenza virus H9N2 for co-infection with Escherichia coli to cause colibacillosis, the importance of lung load of E. coli and the involvement of respiratory phagocytes. 2. Specific pathogen free chickens were inoculated intranasally with 106EID50 of influenza virus or uninfected. After specified time intervals, 107 CFU E. coli or phosphate-buffered saline was inoculated. The presence of lesions, the number of respiratory phagocytes in the respiratory lavage fluid and the E. coli load in the lung were determined after different time intervals. 3. Compared with the number of lesions in chickens receiving only E. coli inoculation, the number lesions in co-infected chickens were increased at 0- and 3-d time intervals, but reduced in the groups at 6- and 9-d intervals between co-infection. 4. At 1-3 d after E. coli inoculation, the number of lesions chickens was correlated with the number of respiratory phagocytes harvested and related to the E. coli load in the lungs at 5 d. 5. These results suggest that the lesions caused by E. coli in chickens were increased within a 0-3 d interval following H9N2 virus inoculation and that this effect is related to the number of respiratory phagocytes.

Mycoplasma gallisepticum modifies the pathogenesis of influenza A virus in the avian tracheal epithelium.

Multiple respiratory infections have a significant impact on health and economy. Pathogenesis of co-infecting viruses and bacteria and their interaction with mucosal surfaces are poorly characterized. In this study we established a co-infection model based on pre-incubation of tracheal organ cultures (TOC) with Mycoplasma (M.) gallisepticum and a subsequent infection with avian influenza virus (AIV). Mycoplasma gallisepticum modified the pathogenesis of AIV as demonstrated in TOC of two different avian species (chickens and turkeys). Co-infection promoted bacterial growth in tracheal epithelium. Depending on the interaction time of M. gallisepticum with the host cells, AIV replication was either promoted or suppressed. M. gallisepticum inhibited the antiviral gene expression and affected AIV attachment to the host cell by desialylation of α-2,3 linked sialic acids. Ultrastructural analysis of co-infected TOC suggests that both pathogens may attach to and possibly infect the same epithelial cell. The obtained results contribute to better understanding of the interaction dynamics between M. gallisepticum and AIV. They highlight the importance of the time interval between infections as well as the biological properties of the involved pathogens as influencing factors in the outcome of respiratory infections.

Patients infected with avian influenza A H7N9 virus have abnormally low thyroid hormone levels.

BACKGROUND: In March 2013, the novel avian influenza A H7N9 virus spread throughout China. This study evaluated whether thyroid function was altered in patients infected with H7N9 virus. METHODS: We analyzed thyroid-stimulating hormone (TSH), total triiodothyronine (TT3), total tetraiodothyronine (TT4), free triiodothyronine (FT3), and free tetraiodothyronine (FT4) levels in 17 patients infected with H7N9 on admission to our hospital and compared the values to the reference ranges for these thyroid hormones. RESULTS: Of the 17 patients, 12 (70.6%) patients had abnormally low TT3 levels, 10 (58.8%) patients had abnormally low FT3 and TSH levels, and 5 (29.4%) patients had abnormally low TT4 and FT4 levels (below the lower limit of the reference ranges for each hormone). CONCLUSIONS: Abnormally low thyroid hormone levels was common in patients infected with H7N9.

Evaluation of the pooling of swabs for real-time PCR detection of low titre shedding of low pathogenicity avian influenza in turkeys.

The purpose of this study was to determine whether pooling avian influenza (AI)-positive swabs with negative swabs has a detrimental effect on the sensitivity of AI real-time reverse transcription-polymerase chain reactions (rRT-PCRs). Cloacal and buccal swabs were sampled daily from 12 turkeys infected with A/goose/England/07(H2N2). For half the turkeys, each swab was mixed with four swabs from known AI-negative turkeys, and for the other half the swabs were tested individually. Bayesian modelling was used to (i) determine whether pooling the positive swabs compromised the cycle threshold (C(t)) value obtained from the rRT-PCRs, and (ii) estimate the likelihood of detection of an H2N2 infected turkey flock via rRT-PCR for pooled and individually tested swabs (cloacal and buccal) vs. the number of days post-infection of the flock. Results indicated that there was no significant effect of compromising AI rRT-PCR sensitivity by pooling a weak positive swab with negative swabs on the Ct values which were obtained. Pooled sampling was able to widen the detection window compared to individual sampling, for the same number of rRT-PCR tests. This indicates that pooled sampling would be an effective method of reducing the number of tests to be performed to determine flock status during an AI outbreak and for surveillance.

Effect of intranasal administration of Lactobacillus fermentum CJL-112 on horizontal transmission of influenza virus in chickens.

The aim of this study was to determine whether intranasal administration of Lactobacillus sp. could prevent horizontal transmission of H9N2 avian influenza virus (AIV) in specific-pathogen-free chickens. Three-week-old chickens received 500 µL of 1.5 × 10(9) cfu of Lactobacillus fermentum CJL-112 strain (CJL) intranasally for 7 d before and 14 d after a challenge. Challenged chickens, each inoculated with H9N2 AIV, were kept in either direct or indirect contact with naive chickens, and morbidity and viral shedding were monitored. We demonstrated that the intranasal administration of CJL significantly decreased the number of chickens with viral shedding from the gastrointestinal tract in the indirect contact chickens (P < 0.001) and also significantly reduced viral shedding from the respiratory tract in the challenged (P < 0.05) and the direct contact chickens (P < 0.001) than those in the control group. Hence, the use of this lactobacilli strain may constitute a novel and effectively plausible alternative to prevent and control H9N2 AIV infection in chickens.

Effect of low-pathogenicity influenza virus H3N8 infection on Mycoplasma gallisepticum infection of chickens.

Mycoplasma infection is still very common in chicken and turkey flocks. Several low-pathogenicity avian influenza (LPAI) viruses are circulating in wild birds that can be easily transmitted to poultry flocks. However, the effect of LPAI on mycoplasma infection is not well understood. The aim of the present study was to investigate the infection of LPAI virus H3N8 (A/mallard/Hungary/19616/07) in chickens challenged with Mycoplasma gallisepticum. Two groups of chickens were aerosol challenged with M. gallisepticum. Later one of these groups and one mycoplasma-free group were aerosol challenged with the LPAI H3N8 virus. The birds were observed for clinical signs for 8 days, then euthanized, and examined for the presence of M. gallisepticum in the trachea, lung, air sac, liver, spleen, kidney and heart, and for developing anti-mycoplasma and anti-viral antibodies. The LPAI H3N8 virus did not cause any clinical signs but M. gallisepticum infection caused clinical signs, reduction of body weight gain and colonization of the inner organs. These parameters were more severe in the birds co-infected with M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone. In addition, in the birds infected with both M. gallisepticum and LPAI H3N8 virus, the anti-mycoplasma antibody response was reduced significantly when compared with the group challenged with M. gallisepticum alone. Co-infection with LPAI H3N8 virus thus enhanced pathogenesis of M. gallisepticum infection significantly.

Distribution patterns of influenza virus receptors and viral attachment patterns in the respiratory and intestinal tracts of seven avian species.

This study assessed the presence of sialic acid α-2,3 and α-2,6 linked glycan receptors in seven avian species. The respiratory and intestinal tracts of the chicken, common quail, red-legged partridge, turkey, golden pheasant, ostrich, and mallard were tested by means of lectin histochemistry, using the lectins Maackia amurensis agglutinin II and Sambucus nigra agglutinin, which show affinity for α-2,3 and α-2,6 receptors, respectively. Additionally, the pattern of virus attachment (PVA) was evaluated with virus histochemistry, using an avian-origin H4N5 virus and a human-origin seasonal H1N1 virus. There was a great variation of receptor distribution among the tissues and avian species studied. Both α-2,3 and α-2,6 receptors were present in the respiratory and intestinal tracts of the chicken, common quail, red-legged partridge, turkey, and golden pheasant. In ostriches, the expression of the receptor was basically restricted to α-2,3 in both the respiratory and intestinal tracts and in mallards the α-2,6 receptors were absent from the intestinal tract. The results obtained with the lectin histochemistry were, in general, in agreement with the PVA. The differential expression and distribution of α-2,3 and α-2,6 receptors among various avian species might reflect a potentially decisive factor in the emergence of new viral strains.

Avian influenza: public health and food safety concerns.

Avian influenza (AI) is a disease or asymptomatic infection caused by Influenzavirus A. AI viruses are species specific and rarely cross the species barrier. However, subtypes H5, H7, and H9 have caused sporadic infections in humans, mostly as a result of direct contact with infected birds. H5N1 high pathogenicity avian influenza (HPAI) virus causes a rapid onset of severe viral pneumonia and is highly fatal (60% mortality). Outbreaks of AI could have a severe economic and social impact on the poultry industry, trade, and public health. Surveillance data revealed that H5N1 HPAI has been detected in imported frozen duck meat from Asia, and on the surface and in contaminated eggs. However, there is no direct evidence that AI viruses can be transmitted to humans via the consumption of contaminated poultry products. Implementing management practices that incorporate biosecurity principles, personal hygiene, and cleaning and disinfection protocols, as well as cooking and processing standards, are effective means of controlling the spread of the AI viruses.

Critical control points for avian influenza A H5N1 in live bird markets in low resource settings.

Live bird markets can become contaminated with and become a source of transmission for avian influenza viruses including the highly pathogenic H5N1 strain. Many countries affected by the H5N1-virus have limited resources for programs in environmental health, sanitation and disease control in live bird markets. This study proposes five critical control points (CCPs) to reduce the risk of H5N1-virus contamination in markets in low resource settings. The CCPs were developed based on three surveys conducted in Indonesia: a cross-sectional survey in 119 markets, a knowledge, attitudes and practice survey in 3 markets and a microbiological survey in 83 markets. These surveys assessed poultry workflow, market infrastructure, hygiene and regulatory practices and microbiological contamination with the H5N1-virus. The five CCPs identified were (1) reducing risk of receiving infected birds into the market, (2) reducing the risk of virus spread between different bird flocks in holding cages, (3) reducing surface contamination by isolating slaughter processes from other poultry-related processes, (4) minimizing the potential for contamination during evisceration of carcasses and (5) reducing the risk of surface contamination in the sale zone of the market. To be relevant for low resource settings, the CCPs do not necessitate large infrastructure changes. The CCPs are suited for markets that slaughter poultry and have capacity for daily disposal and removal of solid waste from the market. However, it is envisaged that the CCPs can be adapted for the development of risk-based programs in various settings.

Qualitative detection of avian influenza A (H5N1) viruses: a comparative evaluation of four real-time nucleic acid amplification methods.

The aim of this study was to determine the performance of real-time amplification based methods - NASBA, TaqMan, RT-FRET, and RT-PCR LUXtrade mark formats - for the detection of influenza A (H5N1) virus RNA. In an analysis of 54 samples obtained from a range of animal species in Thailand during the period 2003-2006, results showed that the NASBA (H5=98.2%, N1=96.3%), TaqMan (H5=98.2%, N1=96.3%) and FRET (H5=98.2%, N1=96.3%) had significantly higher rates of positive detection than LUX (H5=94.4%, N1=50.0%; P<0.001) for influenza A, H5 and N1 isolates. There were no false-positive results from any methods used in the negative-control group of samples. The limits of analytical detection were at least 10copies/reaction in real-time NASBA and LUX assays, while FRET and TaqMan assay appeared to be less sensitive at > or =100copies/reaction. The assays were relatively specific without cross-reactivity to a number of other influenza strains or viral pathogens. In conclusion, our study demonstrated that real-time NASBA, TaqMan and FRET assays can be used to detect influenza A (H5N1) from a wide range of hosts, and be specific for H5N1 samples obtained during different outbreaks (2003-2006). All assays provided the benefit of rapid influenza H5N1 identification for early diagnosis, in the range of hours, and they are well suited to high throughput analyses.

Migratory birds, the H5N1 influenza virus and the scientific method.

BACKGROUND: The role of migratory birds and of poultry trade in the dispersal of highly pathogenic H5N1 is still the topic of intense and controversial debate. In a recent contribution to this journal, Flint argues that the strict application of the scientific method can help to resolve this issue. DISCUSSION: We argue that Flint's identification of the scientific method with null hypothesis testing is misleading and counterproductive. There is far more to science than the testing of hypotheses; not only the justification, bur also the discovery of hypotheses belong to science. We also show why null hypothesis testing is weak and that Bayesian methods are a preferable approach to statistical inference. Furthermore, we criticize the analogy put forward by Flint between involuntary transport of poultry and long-distance migration. SUMMARY: To expect ultimate answers and unequivocal policy guidance from null hypothesis testing puts unrealistic expectations on a flawed approach to statistical inference and on science in general.

La primera pandemia del siglo XXI / The first pandemic disease of XXI century

La gripe aviar es una enfermedad causada por el virus de la influenza tipo A, perteneciente a la familia Orthomyxoviridae, unos 200 millones de aves muertas o sacrificadas y el deceso de 129 personas es el saldo actual de la infección por la misma. Teniendo en cuenta la inminente llegada a nuestro continente y en especial a Cuba, decidimos realizar esta revisión bibliográfica, para ello consultamos las bases de datos CUMED, LILACS, MEDLINE, WHOLIS y el localizador de información de salud LIS Cuba; en la misma se expone de forma actualizada sus principales características microbiológicas, epidemiológicas, clínicas y su situación actual el mundo. Se aborda además sobre la existencia del Sistema de Vigilancia cubano para esta enfermedad y su fortalecimiento, así como la repercusión social, económica y en materia de salud pública que produciría la llegada de lo que ya se considera la posible primera pandemia del presente siglo.