Experimentally induced lameness in turkeys inoculated with a newly emergent turkey reovirus.

Newly emergent turkey arthritis reoviruses (TARVs) have been isolated from cases of lameness in male turkeys over 10 weeks of age. In a previous study, experimental inoculation of TARV in one-week-old turkey poults produced lymphocytic tenosynovitis at four weeks post inoculation but without causing clinical lameness. This study was undertaken to determine if TARV infection at an early age can lead to clinical lameness in birds as they age. One-week-old male turkeys were inoculated orally with a TARV (strain TARV-O'Neil) and monitored for the development of gait defects until 16 weeks of age. At 4, 8, 12 and 16 weeks of age, a subset of birds was euthanized followed by the collection of gastrocnemius tendon, digital flexor tendon, and intestines for virus detection by rRT-PCR and for histologic inflammation scoring. Clinical lameness was first displayed in TARV-infected turkeys at 8 weeks of age and ruptured gastrocnemius tendons with progressive lameness were also seen at 12-16 weeks of age. The virus was detected in gastrocnemius tendon of 4- 8- and 12-week-old turkeys but not in 16-week-old turkeys. Histologic inflammation scores of tendons at each of the four time points were significantly higher in the virus-inoculated group than in the control group (p < 0.01). Lesions began as lymphocytic tenosynovitis with mild synoviocyte hyperplasia at four weeks of age and progressed to fibrosis as the birds aged. These results demonstrate the potential of TARV to infect young turkeys and to produce subclinical tenosynovitis that becomes clinically demonstrable as the turkeys age.

Survival of turkey arthritis reovirus in poultry litter and drinking water.

Turkey reoviruses (TRVs) can cause arthritis, tenosynovitis, and enteric diseases in turkeys, leading to huge economic losses. The TRVs are tentatively divided into turkey arthritis reoviruses (TARVs) and turkey enteric reoviruses (TERVs) depending on the type of disease they produce. This study was conducted to determine the survival of these viruses in autoclaved and nonautoclaved poultry litter and drinking water at room temperature (approx. 25°C). Three isolates of TARV (TARV-O'Neil, TARV-MN2, and TARV-MN4) and one each of TERV (TERV-MN1) and chicken arthritis reovirus (CARV) were used in this study. The viruses were propagated and titrated on QT-35 cells. In autoclaved dechlorinated tap water, all 5 viruses were able to survive for 9 to 13 wk. In nonautoclaved water, all 5 viruses survived for at least 2 wk. In autoclaved litter, the viruses survived for 6 to 8 wk, and in nonautoclaved litter, they survived for 6 to 8 d only. The implications of these results are discussed below.

Prolonged repeated inseminations trigger a local immune response and accelerate aging of the uterovaginal junction in turkey hens.

Artificial insemination is a standard practice in the turkey breeder industry to ensure the production of fertile eggs. Even though hens are inseminated on a weekly basis, their fertility tends to decline after a few weeks of production. Avian species have a specialized structures called sperm storage tubules (SSTs), located in the uterovaginal junction (UVJ) of the oviduct. The ability of SSTs to store sperm is directly correlated with the fertility of the hen. The objective of the study was to examine changes in the transcriptome of the turkey hen's UVJ in response to the presence of sperm at three key stages of production. We hypothesized that repeated and prolonged exposure to sperm would alter the transcriptome of the UVJ. Samples were collected from virgin hens prior to the onset of lay, as well as from sham-inseminated (extender only) and semen-inseminated hens at early lay, peak lay, and late lay. Gene expression profiling of the UVJ was examined, and a differential expression analysis was conducted through pairwise comparisons between semen- and sham-inseminated groups at each production stage and across production stages. In the early laying stage, no significant gene expression changes were found between semen- and sham-inseminated groups. However, at peak lay, genes related to lipid biosynthesis, Wnt signaling, cell proliferation, and O-glycan biosynthesis were upregulated in the semen group, while the immune response and cytokine-cytokine receptor interaction were downregulated. In the late lay stage, the transcription pathway was upregulated in the semen group, whereas the translation pathway was downregulated. The local immune response that was suppressed during peak lay was increased at the late laying stage. In the semen-inseminated group, the UVJ exhibited advanced aging at the late laying stage, evidenced by reduced telomere maintenance and translation processes. The results from this study provide valuable insights into the alteration of the UVJ function in response to the presence of sperm at different stages of production and throughout the production cycle. Targeting the modulation of local immune response and addressing aging processes after peak production could potentially prevent or delay the decline in fertility of turkey breeder hens.

Antibiotics and Host-Tailored Probiotics Similarly Modulate Effects on the Developing Avian Microbiome, Mycobiome, and Host Gene Expression.

The microbiome is important to all animals, including poultry, playing a critical role in health and performance. Low-dose antibiotics have historically been used to modulate food production animals and their microbiome. Identifying alternatives to antibiotics conferring similar modulatory properties has been elusive. The purpose of this study was to determine if a host-tailored probiotic could recapitulate effects of a low-dose antibiotic on host response and the developing microbiome. Over 13 days of life, turkey poults were supplemented continuously with a low-dose antibiotic or oral supplementation of a prebiotic with or without two different probiotics (8 cage units, n = 80 per group). Gastrointestinal bacterial and fungal communities of poults were characterized by 16S rRNA gene and ITS2 amplicon sequencing. Localized and systemic host gene expression was assessed using transcriptome sequencing (RNA-Seq), kinase activity was assessed by avian-specific kinome peptide arrays, and performance parameters were assessed. We found that development of the early-life microbiome of turkey poults was tightly ordered in a tissue- and time-specific manner. Low-dose antibiotic and turkey-tailored probiotic supplementation, but not nontailored probiotic supplementation, elicited similar shifts in overall microbiome composition during development compared to controls. Treatment-induced bacterial changes were accompanied by parallel shifts in the fungal community and host gene expression and enhanced performance metrics. These results were validated in pen trials that identified further additive effects of the turkey-tailored probiotic combined with different prebiotics. Alternative approaches to low-dose antibiotic use in poultry are feasible and can be optimized utilizing the indigenous poultry microbiome. Similar approaches may also be beneficial for humans.IMPORTANCE Alternative approaches are greatly needed to reduce the need for antibiotic use in food animal production. This study utilized a pipeline for the development of a host-tailored probiotic to enhance performance in commercial turkeys and modulate their microbiota, similar to the effects of low-dose antibiotic administration. We determined that a host-tailored probiotic, developed in the context of the commercial turkey gut microbiome, was more effective at modulating these parameters than a nontailored probiotic cocktail. Furthermore, the host-tailored probiotic mimicked many of the effects of a low-dose antibiotic growth promoter. Surprisingly, the effects of the antibiotic growth promoter and host-tailored probiotic were observed across kingdoms, illustrating the coordinated interkingdom effects of these approaches. This work suggests that tailored approaches to probiotic development hold promise for modulating the avian host and its microbiota.

Comparative pathogenicity of early and recent isolates of avian metapneumovirus subtype C in turkeys.

The objective of the present study was to compare the pathogenicity of early and recent isolates of avian metapneumovirus subtype-C (aMPV-C) in turkeys. Two-week-old turkeys were inoculated with early and recent isolates of aMPV-C. Clinical signs were monitored. Tissues were examined for viral ribonucleic acid (RNA), lesions, and viral antigen by reverse transcription-polymerase chain reaction (RT-PCR), histopathology and immunohistochemistry, respectively. Birds infected with the recent isolate had higher clinical sign scores than those infected with the early isolate. Only the recent isolate produced a multifocal loss of cilia in the nasal turbinate of infected birds. Immunohistochemistry revealed intense staining of aMPV antigen in turbinate and trachea of birds infected with the recent isolate. The findings indicate that the recent isolate produced more severe clinical signs and lesions in turkeys compared to the early isolate. The recent isolate could be ideal for the development of a challenge model for aMPV infection in turkeys.

Rapid detection of avian pneumovirus in tissue culture by microindirect immunofluorescence test.

An indirect immunofluorescence (IFA) test with a 96-well, flat-bottomed microplate was developed to detect avian pneumovirus (APV) antigen in Vero cell cultures. Samples of nasal turbinates and swabs from infraorbital sinuses and trachea were collected from 4-week-old poults experimentally inoculated with APV. The APV titers by tissue culture IFA staining were compared with that of visual reading of cytopathic effect (CPE). The ability of IFA staining to detect APV antigen correlated well with visualizing CPE. The use of IFA staining of Vero cell cultures allowed detection of APV in substantially less time than the use of visualizing CPE. In addition, the use of IFA allowed specific identification of the virus in cell culture.

Avian pneumovirus and its survival in poultry litter.

The survival of avian pneumovirus (APV) in turkey litter was studied at different temperature (room temperature, [approximately 22-25 C], 8 C, and -12 C) conditions. Built-up turkey litter from a turkey breeder farm known to be free of APV was obtained and was divided into two portions. One portion was sterilized by autoclaving and the other portion was kept nonautoclaved. Both samples were inoculated with a Vero cell-propagated Minnesota isolate of APV subtype C (APV/MN2A) with a titer of 10(5) 50% tissue culture infective dose at 1% level. These samples were then stored at three different temperatures: -12 C, 8 C, and room temperature (20-25 C). The samples were tested for the presence of viral RNA by reverse transcriptase-polymerase chain reaction and for the presence of live virus by virus isolation in Vero cells at the intervals of 1, 2, 3, 7, 14, 30, 60, and 90 days. Our studies revealed the presence of APV RNA even after 90 days in the autoclaved litter samples kept at -12 C and at 8 C. The virus was isolated from the autoclaved litter kept at -12 C up to 60 days. From the nonautoclaved litter, viral RNA was detected up to 60 days and virus was isolated up to 14days. The present study indicated that APV could survive in built-up turkey litter up to 60 days postinoculation at a temperature of-12 C.

Effects of bacterial coinfection on the pathogenesis of avian pneumovirus infection in turkeys.

Four- and nine-week-old poults were inoculated with cell culture propagated avian pneumovirus (APV) into each conjunctival space and nostril, followed by inoculation 3 days later with Escherichia coli, Bordetella avium (BA), or Ornithobacterium rhinotracheale or a mixture of all three (EBO). Clinical signs were evaluated on days 3, 5, 7, 9, 11, and 14 postinoculation (PI) of APV. The poults were euthanatized on days 2, 4, 6, 10, and 14 PI, and blood and tissues were collected. The poults that received APV followed by EBO or BA alone developed more severe clinical signs related to nasal discharge and swelling of intraorbital sinuses than did poults inoculated with APV alone or bacteria alone. More severe pathologic changes were found in poults inoculated with APV+BA that extended to the air sacs and lungs, particularly in 9-wk-old poults. Bordetella avium was recovered from tracheas and lungs of birds that were inoculated with APV followed by EBO or BA alone. APV was detected by immunohistochemical staining in the upper respiratory tract longer in the groups of poults inoculated with APV and pathogenic bacteria than in those that received only APV, particularly when BA was involved. Viral antigen was also detected in the lungs of poults that were inoculated with APV followed by administration of EBO or BA alone. Loss of cilia on the epithelial surface of the upper respiratory tract was associated with BA infection and may enhance infection with APV, allowing deeper penetration of the virus into the respiratory tract.

Conversion of distiller's grain into fuel alcohol and a higher-value animal feed by dilute-acid pretreatment.

Over the past three decades ethanol production in the United States has increased more than 10-fold, to approx 2.9 billion gal/yr (mid-2003), with ethanol production expected to reach 5 billion gal/yr by 2005. The simultaneous coproduction of 7 million t/yr of distiller's grain (DG) may potentially drive down the price of DG as a cattle feed supplement. The sale of residual DG for animal feed is an important part of corn dry-grind ethanol production economics; therefore, dry-grind ethanol producers are seeking ways to improve the quality of DG to increase market penetration and help stabilize prices. One possible improvement is to increase the protein content of DG by converting the residual starch and fiber into ethanol. We have developed methods for steam explosion, SO2, and dilute-sulfuric acid pretreatment of DG for evaluation as a feedstock for ethanol production. The highest soluble sugar yields (approximately 77% of available carbohydrate) were obtained by pretreatment of DG at 140 degrees C for 20 min with 3.27 wt% H2SO4. Fermentation protocols for pretreated DG were developed at the bench scale and scaled to a working volume of 809 L for production of hydrolyzed distiller's grain (HDG) for feeding trials. The pretreated DG was fermented with Saccharomyces cerevisiae D5A, with ethanol yields of 73% of theoretical from available glucans. The HDG was air-dried and used for turkey-feeding trials. The inclusion of HDG into turkey poult (as a model non-ruminant animal) diets at 5 and 10% levels, replacing corn and soybean meal, showed weight gains in the birds similar to controls, whereas 15 and 20% inclusion levels showed slight decreases (-6%) in weight gain. At the conclusion of the trial, no negative effects on internal organs or morphology, and no mortality among the poults, was found. The high protein levels (58-61%) available in HDG show promising economics for incorporation of this process into corn dry-grind ethanol plants.

Succession of the turkey gastrointestinal bacterial microbiome related to weight gain.

Because of concerns related to the use of antibiotics in animal agriculture, antibiotic-free alternatives are greatly needed to prevent disease and promote animal growth. One of the current challenges facing commercial turkey production in Minnesota is difficulty obtaining flock average weights typical of the industry standard, and this condition has been coined "Light Turkey Syndrome" or LTS. This condition has been identified in Minnesota turkey flocks for at least five years, and it has been observed that average flock body weights never approach their genetic potential. However, a single causative agent responsible for these weight reductions has not been identified despite numerous efforts to do so. The purpose of this study was to identify the bacterial community composition within the small intestines of heavy and light turkey flocks using 16S rRNA sequencing, and to identify possible correlations between microbiome and average flock weight. This study also sought to define the temporal succession of bacteria occurring in the turkey ileum. Based upon 2.7 million sequences across nine different turkey flocks, dominant operational taxonomic units (OTUs) were identified and compared between the flocks studied. OTUs that were associated with heavier weight flocks included those with similarity to Candidatus division Arthromitus and Clostridium bartlettii, while these flocks had decreased counts of several Lactobacillus species compared to lighter weight flocks. The core bacterial microbiome succession in commercial turkeys was also defined. Several defining markers of microbiome succession were identified, including the presence or abundance of Candidatus division Arthromitus, Lactobacillus aviarius, Lactobacillus ingluviei, Lactobacillus salivarius, and Clostridium bartlettii. Overall, the succession of the ileum bacterial microbiome in commercial turkeys proceeds in a predictable manner. Efforts to prevent disease and promote growth in the absence of antibiotics could involve target dominant bacteria identified in the turkey ileum that are associated with increased weight gain.

Development of a vaccine-challenge model for avian metapneumovirus subtype C in turkeys.

The objective of this study was to evaluate different preparations of avian metapneumovirus (aMPV) subtype C as vaccine challenge in turkeys. Two aMPV isolates and their respective nasal turbinate homogenates after propagation in turkeys were used in the study. Significantly higher clinical sign scores were recorded in birds inoculated with 20 or 2% turbinate homogenate of recent isolate. Birds in the above groups showed more pronounced histopathological lesions, and a higher percentage of birds showed viral RNA and antigen in tissues. The data demonstrated that nasal turbinate homogenate of recent isolate produced severe clinical signs and lesions in turkeys and could be an ideal candidate for vaccine-challenge studies.