Consumption of lysozyme-rich milk can alter microbial fecal populations.

Human milk contains antimicrobial factors such as lysozyme and lactoferrin that are thought to contribute to the development of an intestinal microbiota beneficial to host health. However, these factors are lacking in the milk of dairy animals. Here we report the establishment of an animal model to allow the dissection of the role of milk components in gut microbiota modulation and subsequent changes in overall and intestinal health. Using milk from transgenic goats expressing human lysozyme at 68%, the level found in human milk and young pigs as feeding subjects, the fecal microbiota was analyzed over time using 16S rRNA gene sequencing and the G2 Phylochip. The two methods yielded similar results, with the G2 Phylochip giving more comprehensive information by detecting more OTUs. Total community populations remained similar within the feeding groups, and community member diversity was changed significantly upon consumption of lysozyme milk. Levels of Firmicutes (Clostridia) declined whereas those of Bacteroidetes increased over time in response to the consumption of lysozyme-rich milk. The proportions of these major phyla were significantly different (P < 0.05) from the proportions seen with control-fed animals after 14 days of feeding. Within phyla, the abundance of bacteria associated with gut health (Bifidobacteriaceae and Lactobacillaceae) increased and the abundance of those associated with disease (Mycobacteriaceae, Streptococcaceae, Campylobacterales) decreased with consumption of lysozyme milk. This study demonstrated that a single component of the diet with bioactivity changed the gut microbiome composition. Additionally, this model enabled the direct examination of the impact of lysozyme on beneficial microbe enrichment versus detrimental microbe reduction in the gut microbiome community.

Prevalence of low pathogenicity avian influenza virus during 2005 in two U.S. live bird market systems.

Oropharyngeal and cloacal swabs were collected from poultry sold in two live bird market (LBM) systems to estimate the prevalence of low pathogenicity avian influenza virus (LPAIV) shedding during the summer and fall of 2005. Random sampling was conducted in three LBMs in Minnesota where 50 birds were sampled twice weekly for 4 wk, and in three LBMs in a California marketing system. A stratified systematic sampling method was used to collect samples from Southern California LBMs, where LPAIV was detected during routine surveillance. No LPAIV was detected in the LBM system in Minnesota where realtime reverse transcription-PCR (RT-PCR) was conducted on oropharyngeal samples. RT-PCR was performed on swabs taken from 290 of 14,000, 65 of 252, and 60 of 211 birds at the three Southern California LBMs. The number of samples collected was based on the number of birds, age of the birds, and number of species present in the LBM. Virus isolation, subtyping, and sequencing of the hemagglutinin, neuraminidase, and other internal protein genes was performed on AIV-positive samples. The estimated prevalence of LPAIV in California was 0.345% in an LBM/supply farm with multiple ages of Japanese quail, 3% in an LBM with multiple ages and strains of chickens present, and 49.8% in an LBM with multiple species, multiple strains, and multiple ages. The positive virus samples were all LPAIV H6N2 and closely related to viruses isolated from Southern California in 2001 and 2004. Little or no comingling of poultry may contribute to little or no LPAIV detection in the LBMs.

Roles of the ERK MAPK in the regulation of proinflammatory and apoptotic responses in chicken macrophages infected with H9N2 avian influenza virus.

The mitogen-activated protein kinase (MAPK) family is responsible for important signalling pathways which regulate cell activation, differentiation, apoptosis and immune responses. Studies have shown that influenza virus infection activates MAPK family members in mammals. While the extracellular signal-regulated kinase (ERK)1/2 is important for virus replication, activation of p38 controls the expression of RANTES, interleukin (IL)-8 and tumour necrosis factor (TNF)-alpha. In this study, we report that avian influenza virus (AIV) activates ERK, p38 and Jun-N-terminal kinases in avian species. In chicken macrophages, while ERK was required for H9N2 AIV replication, ERK regulated proinflammatory cytokines IL-1beta, IL-6 and IL-8, which is distinct from what has been previously reported in mammalian cells. Moreover, ERK alone suppressed TNF-alpha and FasL and inhibited TNF-family-mediated extrinsic apoptosis in H9N2-infected chicken macrophages. Taken together, these findings suggest that ERK signalling may uniquely play important roles in avian host responses to AIV infection.

High-throughput neuraminidase substrate specificity study of human and avian influenza A viruses.

Despite the importance of neuraminidase (NA) activity in effective infection by influenza A viruses, limited information exists about the differences of substrate preferences of viral neuraminidases from different hosts or from different strains. Using a high-throughput screening format and a library of twenty α2-3- or α2-6-linked para-nitrophenol-tagged sialylgalactosides, substrate specificity of NAs on thirty-seven strains of human and avian influenza A viruses was studied using intact viral particles. Neuraminidases of all viruses tested cleaved both α2-3- and α2-6-linked sialosides but preferred α2-3-linked ones and the activity was dependent on the terminal sialic acid structure. In contrast to NAs of other subtypes of influenza A viruses which did not cleave 2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid (Kdn) or 5-deoxy Kdn (5d-Kdn), NAs of all N7 subtype viruses tested had noticeable hydrolytic activities on α2-3-linked sialosides containing Kdn or 5d-Kdn. Additionally, group 1 NAs showed efficient activity in cleaving N-azidoacetylneuraminic acid from α2-3-linked sialoside.

Adaptation and transmission of a duck-origin avian influenza virus in poultry species.

A duck-origin avian influenza virus (AIV) was used to study viral adaptation and transmission patterns in chickens (Gallus gallus domesticus) and Pekin ducks (Anas platyrhynchos domesticus). Inoculated birds were housed with naïve birds of the same species and all birds were monitored for infection. The inoculating duck virus was transmitted effectively by contact in both species. Viruses recovered from infected birds showed mutations as early as 1 or 3 days after inoculation in chickens and ducks, respectively. Amino acid substitutions in hemagglutinin (HA) or deletions in neuraminidase (NA) stalk regions were identified in chicken isolates, but only substitutions in HA were identified in duck isolates. HA substitution-containing viruses replicated more efficiently than those with NA stalk deletions. NA deletion mutants were not recovered from contact chickens, suggesting inefficient transmission. Amino acid substitutions in HA proteins appeared in pairs in chickens, but were independent in ducks, indicating adaptation in chickens. In addition, our findings showed that a duck-origin virus can rapidly adapt to chickens, suggesting that the emergence of new epidemic AIV can be rapid.

Influenza A viruses in wild birds of the Pacific flyway, 2005-2008.

Avian influenza viruses (AIVs) pose a significant threat to public health, and viral subtypes circulating in natural avian reservoirs can contribute to the emergence of pathogenic influenza viruses in humans. We investigated the prevalence and distribution of AIVs in 8826 migratory and resident wild birds in North America along the Pacific flyway, which is a major north-south migration pathway that overlaps with four other flyways in Alaska providing opportunities for mixing of Eurasian and American origin influenza viruses. Overall, the prevalence of AIVs was low (1%) among the wide range of avian species tested, but we detected AIVs in 69 hunter-harvested waterfowl (Anseriformes) sampled at a national wildlife refuge in California from October 2007 to January 2008. A wide range of subtypes were detected in waterfowl with H6N1, H10N7, H7N3, and H3N5 being the most common. We suspect H6N1 was introduced or remerged in 2007 at this key wintering site for waterfowl along the Pacific Flyway. Over a 3-week period, 13 H6N1 AIVs were isolated from two northern pintails (Anas acuta), three northern shovelers (Anas clypeata), three ring-necked ducks (Aythya collaris), four American widgeon (Anas americana), and one gadwall (Anas strepera). We conclude that a diverse array of AIVs was present and that cross-species transmission was occurring among waterfowl in the central valley wetlands of California.

Modulation of the immune responses in chickens by low-pathogenicity avian influenza virus H9N2.

Most low-pathogenicity avian influenza (LPAI) viruses cause no or mild disease in avian species. Little is known about the mechanisms of host defence and the immune responses of avian influenza-infected birds. This study showed that chicken macrophages are susceptible to infection with LPAI H9N2 and H6N2 viruses and that infection led to apoptosis. In H9N2 virus-infected chicken macrophages, Toll-like receptor 7 responded to infection and mediated the cytokine responses. Whilst pro-inflammatory cytokines were largely upregulated, the interferon (IFN) response was fairly weak and IFN-inducible genes were differentially regulated. Among the regulated genes, major histocompatibility complex (MHC) antigens II were downregulated, which also occurred in the lungs of H9N2-infected chickens. Additionally, interleukin (IL)-4, IL-4 receptor and CD74 (MHC class II invariable chain) were also downregulated, all of which are pivotal in the activation of CD4+ helper T cells and humoral immunity. Remarkably, in H9N2 virus-infected chickens, the antibody response was severely suppressed. This was in contrast to the robust antibody response in chickens infected with H6N2 virus, in which expression of MHC class II antigens was upregulated. These data suggest that neutralizing antibodies and humoral immunity may not be developed efficiently in H9N2-infected chickens. These findings raise questions about how some LPAI viruses differentially regulate avian immune responses and whether they have similar effects on mammalian immune function.