Matthew D. Koci

Molecular Characterization of an Avian Astrovirus

ABSTRACT Astroviruses are known to cause enteric disease in several animal species, including turkeys. However, only human astroviruses have been well characterized at the nucleotide level. Herein we report the nucleotide sequence, genomic organization, and predicted amino acid sequence of a turkey astrovirus isolated from poults with an emerging enteric disease.

Genomic Analysis of Closely Related Astroviruses

ABSTRACT To understand astrovirus biology, it is essential to understand factors associated with its evolution. The current study reports the genomic sequences of nine novel turkey astrovirus (TAstV) type 2-like clinical isolates. This represents, to our knowledge, the largest genomic-length data set available for any one astrovirus type. The comparison of these TAstV sequences suggests that the TAstV species contains multiple subtypes and that recombination events have occurred across the astrovirus genome. In addition, the analysis of the capsid gene demonstrated evidence for both site-specific positive selection and purifying selection.

Inactivation of an astrovirus associated with poult enteritis mortality syndrome.

Outbreaks of poult enteritis mortality syndrome (PEMS) continue to cause financial losses to the turkey industry. Clinically, PEMS is defined by mortality profiles, diarrhea, flock unevenness, and immunosuppression. PEMS is a very difficult disease to control and prevent. Depopulation of PEMS-affected flocks and thorough cleaning of the contaminated housing have failed to prevent infection (disease) in subsequent flock placements. The relationship of PEMS to other enteric disease complexes of young turkeys is unknown, partly because the causative agent of PEMS remains unknown. Recently, we isolated a unique astrovirus strain from the thymus and intestines of PEMS-infected poults. This strain is molecularly and serologically distinct from the astrovirus that circulated in turkeys in the 1980s. Mammalian astroviruses are very resistant to inactivation. In these studies, we examined the stability of partially purified PEMS-associated astrovirus to inactivation with heat, laboratory disinfectants, and commercial disinfectants used in commercial turkey houses in an embryonated egg model system. Similar to mammalian astroviruses, the PEMS-associated astrovirus is resistant to inactivation by heat, acidification, detergent treatment, and treatment with phenolic, quaternary ammonium, or benzalkonium chloride-based products. Only treatment with formaldehyde, beta-propriolactone, or the peroxymonosulfate-based product Virkon S completely inactivated the astrovirus in the embryo model. These studies provide an alternate means to potentially control at least one virus associated with PEMS through the use of specific disinfectants.

Development of a Multiplex Reverse Transcription–Polymerase Chain Reaction Diagnostic Test Specific for Turkey Astrovirus and Coronavirus

Abstract A multiplex reverse transcription–polymerase chain reaction (RT-PCR) assay was developedfor the simultaneous detection of two enteric viruses of poultry: turkey enteric coronavirus (TCV) and turkey astrovirus (TAstV). PCR primers were designed to conserved regions within the nucleocapsid gene of TCV and to the polymerase gene of TAstV-2. The primer pairs were successfully used in a multiplex RT-PCR to detect nucleic acid of TAstV-2 and TCV. The test was optimized for use with intestines/feces from naturally infected turkeys. The primers were specific and did not amplify other common RNA or DNA avian viruses. The detection limit was determined to be 10 ng of RNA used as starting template. The use of this specific test allows the rapid and early diagnosis of two financially costly viruses affecting the commercial turkey industry.

Astrovirus-Induced Synthesis of Nitric Oxide Contributes to Virus Control during Infection

ABSTRACT Astrovirus is one of the major causes of infant and childhood diarrhea worldwide. Our understanding of astrovirus pathogenesis trails behind our knowledge of its molecular and epidemiologic properties. Using a recently developed small-animal model, we investigated the mechanisms by which astrovirus induces diarrhea and the role of both the adaptive and innate immune responses to turkey astrovirus type-2 (TAstV-2) infection. Astrovirus-infected animals were analyzed for changes in total lymphocyte populations, alterations in CD4+/CD8+ ratios, production of virus-specific antibodies (Abs), and macrophage activation. There were no changes in the numbers of circulating or splenic lymphocytes or in CD4+/CD8+ ratios compared to controls. Additionally, there was only a modest production of virus-specific Abs. However, adherent spleen cells from infected animals produced more nitric oxide (NO) in response to ex vivo stimulation with lipopolysaccharide. In vitro analysis demonstrated that TAstV-2 induced macrophage production of inducible nitric oxide synthase. Studies using NO donors and inhibitors in vivo demonstrated, for the first time, that NO inhibited astrovirus replication. These studies suggest that NO is important in limiting astrovirus replication and are the first, to our knowledge, to describe the potential role of innate immunity in astrovirus infection.

Development of the Chick Microbiome: How Early Exposure Influences Future Microbial Diversity.

The concept of improving animal health through improved gut health has existed in food animal production for decades; however, only recently have we had the tools to identify microbes in the intestine associated with improved performance. Currently, little is known about how the avian microbiome develops or the factors that affect its composition. To begin to address this knowledge gap, the present study assessed the development of the cecal microbiome in chicks from hatch to 28 days of age with and without a live Salmonella vaccine and/or probiotic supplement; both are products intended to promote gut health. The microbiome of growing chicks develops rapidly from days 1–3, and the microbiome is primarily Enterobacteriaceae, but Firmicutes increase in abundance and taxonomic diversity starting around day 7. As the microbiome continues to develop, the influence of the treatments becomes stronger. Predicted metagenomic content suggests that, functionally, treatment may stimulate more differences at day 14, despite the strong taxonomic differences at day 28. These results demonstrate that these live microbial treatments do impact the development of the bacterial taxa found in the growing chicks; however, additional experiments are needed to understand the biochemical and functional consequences of these alterations.

Astrovirus infection induces sodium malabsorption and redistributes sodium hydrogen exchanger expression.

Astroviruses are known to be a leading cause of diarrhea in infants and the immunocompromised; however, our understanding of this endemic pathogen is limited. Histological analyses of astrovirus pathogenesis demonstrate clinical disease is not associated with changes to intestinal architecture, inflammation, or cell death. Recent studies in vitro have suggested that astroviruses induce actin rearrangement leading to loss of barrier function. The current study used the type-2 turkey astrovirus (TAstV-2) and turkey poult model of astrovirus disease to examine how astrovirus infection affects the ultrastructure and electrophysiology of the intestinal epithelium. These data demonstrate that infection results in changes to the epithelial ultrastructure, rearrangement of F-actin, decreased absorption of sodium, as well as redistribution of the sodium/hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm. Collectively, these data suggest astrovirus infection induces sodium malabsorption, possibly through redistribution of specific sodium transporters, which results in the development of an osmotic diarrhea.

A mastoparan-derived peptide has broad-spectrum antiviral activity against enveloped viruses

Broad-spectrum antiviral drugs are urgently needed to treat individuals infected with new and re-emerging viruses, or with viruses that have developed resistance to antiviral therapies. Mammalian natural host defense peptides (mNHP) are short, usually cationic, peptides that have direct antimicrobial activity, and which in some instances activate cell-mediated antiviral immune responses. Although mNHP have potent activity in vitro, efficacy trials in vivo of exogenously provided mNHP have been largely disappointing, and no mNHP are currently licensed for human use. Mastoparan is an invertebrate host defense peptide that penetrates lipid bilayers, and we reasoned that a mastoparan analog might interact with the lipid component of virus membranes and thereby reduce infectivity of enveloped viruses. Our objective was to determine whether mastoparan-derived peptide MP7-NH2 could inactivate viruses of multiple types, and whether it could stimulate cell-mediated antiviral activity. We found that MP7-NH2 potently inactivated a range of enveloped viruses. Consistent with our proposed mechanism of action, MP7-NH2 was not efficacious against a non-enveloped virus. Pre-treatment of cells with MP7-NH2 did not reduce the amount of virus recovered after infection, which suggested that the primary mechanism of action in vitro was direct inactivation of virus by MP7-NH2. These results demonstrate for the first time that a mastoparan derivative has broad-spectrum antiviral activity in vitro and suggest that further investigation of the antiviral properties of mastoparan peptides in vivo is warranted.

Maternal antibody transfer to broiler progeny varies among strains and is affected by grain source and cage density

Two experiments were conducted to examine the effects of broiler breeder dietary grain source and cage density on maternal antibody (MatAb) transfer to progeny in 2 genetic strains (A and B). Broiler breeders were assigned to 16 litter floor pens and fed either corn- or wheat-based diets. Breeders were administered 4 live vaccines against Newcastle disease virus (NDV). At 23 wk of age, pullets and cocks, which reflected the full BW distribution from each treatment, were moved to a cage breeder house and placed at 1 or 2 hens/cage. Breeders were artificially inseminated at 44 wk (experiment 1) and 52 wk of age (experiment 2). Eggs were collected for 8 d, incubated, and placed in individual pedigree bags at d 19 of incubation. Blood samples from 5 chicks per treatment combination were collected at hatch in both experiments. Spleen and bursa were collected from the same chicks for histomorphometry analyses in experiment 2. In the second experiment, 12 chicks per treatment were placed in cages. Progeny were provided diets based on the same grain (corn or wheat) as their parents. Serum samples were collected at 5, 9, and 13 d of age and analyzed for anti-NDV MatAb. Data were analyzed as a 2 × 2 × 2 factorial design considering strain, dietary grain source, and cage density as main factors. Interaction effects were observed in breeders and progeny. Experiment 1 showed that strain A chicks had lower levels of MatAb when hens were housed at 2 hens/cage rather than 1 hen/cage. The MatAb levels of strain B chickens were not affected by cage density in either experiment. Experiment 2 demonstrated similar effects of cage density on MatAb levels and the area of bursa follicles for both strains. Progeny of breeders fed corn-based diets had smaller spleen white pulp only when hens were housed at 2 hens/cage compared with 1 hen/cage. The results of these experiments suggest that breeder strain and cage-density conditions affected MatAb transfer to progeny and embryo development of spleen and bursa.

Direct fed microbial supplementation repartitions host energy to the immune system.

Direct fed microbials and probiotics are used to promote health in livestock and poultry; however, their mechanism of action is still poorly understood. We previously reported that direct fed microbial supplementation in young broilers reduced ileal respiration without changing whole-body energy expenditure. The current studies were conducted to further investigate the effects of a direct fed microbial on energy metabolism in different tissues of broilers. One hundred ninety-two 1-d-old broiler chicks (16 chicks/pen) were randomly assigned to 2 dietary groups: standard control starter diet (CSD) and CSD plus direct fed microbial (DFMD; 0.3%) with 6 pens/treatment. Body weight, feed consumption, whole-body energy expenditure, organ mass, tissue respiration rates, and peripheral blood mononuclear cell (PBMC) ATP concentrations were measured to estimate changes in energy metabolism. No differences in whole body energy expenditure or BW gain were observed; however, decreased ileal O(2) respiration (P < 0.05) was measured in DFMD fed broilers. In contrast, the respiration rate of the thymus in those broilers was increased (P < 0.05). The PBMC from DFMD fed broilers had increased ATP concentrations and exhibited increased ATP turnover (P < 0.01). To determine if the increased energy consumption by PBMC corresponded with an altered immune response, broilers were immunized with sheep red blood cells (SRBC) and assayed for differences in their humoral response. The DFMD-fed broilers had a faster rate of antigen specific IgG production (P < 0.05) and an increase in total IgA (P < 0.05). Collectively, these data indicate that supplementation with the direct fed microbial used in this study resulted in energy re-partitioning to the immune system and an increase in antibody production independent of changes in whole body metabolism or growth performance.