Donald L. Reynolds

Astrovirus Infection in Hatchling Turkeys: Alterations in Intestinal Maltase Activity

Two experiments were conducted to determine intestinal disaccharidase activity in 1-day-old commercial turkey poults inoculated with astrovirus. Small intestinal samples were collected on days 0.5, 1, 3, and 7 postinoculation (PI) in Expt. 1 and on days 7, 10, and 14 PI in Expt. 2 and evaluated for specific maltase activity (SMA). Astrovirus infection was verified on day 7 PI by immune electron microscopy of intestinal contents. Inoculated poults developed diarrhea and a transient, significant decrease in intestinal SMA. SMA was significantly (P < 0.05) lower in astrovirus-inoculated poults than in control poults throughout the entire small intestine from day 3 through day 7 PI. However, SMA had returned to normal in inoculated poults by day 10 PI and was significantly higher than control values (P < 0.05) in all sections of the small intestine, except in the proximal jejunum, by day 14 PI. Decreased SMA caused by astrovirus infection resulted in disaccharide maldigestion, malabsorption, and subsequent osmotic diarrhea. As astrovirus was cleared from the intestinal tract, SMA was restored and diarrhea was resolved.

Astrovirus infection in hatchling turkeys: histologic, morphometric, and ultrastructural findings.

In three separate experiments, 2- or 5-day-old commercial turkey poults were inoculated orally with astrovirus and examined for clinical signs and for gross and microscopic lesions over a period of 14 days. By day 2 postinoculation (PI), inoculated poults had developed diarrhea, generalized loss of intestinal tone, and dilated ceca that contained light-yellow fluid feces and gas; these changes persisted through day 10 PI. Mild crypt hyperplasia was the only change discernible by light microscopy, and it was first noted in the proximal jejunum on day 1 PI, in the distal jejunum and ileum on day 3 PI, and in the duodenum on day 5 PI. A significant (P < 0.05) increase in crypt depth and area was documented by image analysis on day 3 PI. Ultrastructural evaluation revealed intracytoplasmic aggregates of astrovirus in enterocytes on the sides and base of villi in the ileum and distal jejunum on day 3 PI. Based on the findings, it was concluded that astrovirus caused lesions and replicated in both upper and lower segments of the small intestine in turkey poults.

Environmental Impacts & Bio-security of Composting for Emergency Disposal of Livestock Mortalities

Carcass degradation rate, environmental impacts, and bio-security of windrow-type composting test units were monitored in replicated seasonal trials to assess the feasibility of using composting for emergency disposal of cattle and other large livestock carcasses. Internal temperatures were highest in test units constructed with corn silage. Test units constructed with ground cornstalks or straw and manure were generally 10-20 °C cooler. O2 concentrations in the core of ground cornstalk test units typically exceeded 15%, while those in corn silage and straw/manure test units were in the 5-10% range during the initial weeks of the trials. Despite differences in core temperature and O2 concentration, soft tissue degradation rates were the same in all test units, taking 4-6 months in units constructed during warm weather, and 8-10 months during cold-weather. It is believed that the less favorable (lower) temperatures in the cornstalks may have been offset by significantly higher O2 concentrations which favor rapid aerobic decomposition. Thirty to 45 cm of cover material proved effective in absorbing and retaining odorous gases and leachate. Odors samples collected from the surface of the mortality composting piles typically had low threshold values (< 1500) that differed little from odors emitted by stockpiles of the cover material alone. Leachate volumes were <2% of the precipitation falling on the test units, and preliminary analyses of 1.2 m soil cores show only slight increases in total C and N concentrations in the top 45 cm. Biosecurity tests indicated that pathogens were effectively retained and inactivated: vaccine strains of two avian viruses were inactivated in <21 days; and <2% of sentinel poultry located near the test units exhibited an immune system response to these viruses.

Stunting Syndrome in Turkey Poults: Isolation and Identification of the Etiologic Agent

Stunting syndrome (SS) is an enteric disease of turkey poults that causes high morbidity including reduced growth, impaired feed efficiency, and diarrhea. The etiologic agent of this disease has not been previously reported. The objectives of the present study were to identify, isolate, and purify the etiologic agent of SS. Day-old poults were orally inoculated with a SS-inducing inoculum. The intestinal epithelial cells (IECs) were isolated on the fourth day postinoculation. The IECs were lysed and filtered through 0.2-, 0.1-, and 0.02-micron filters. The cell lysate filtrate (0.1 micron) was subjected to density gradient ultracentrifugation. Intact IECs, filtrates from IECs (0.2, 0.1, and 0.02 micron ), and IEC lysate fractions from gradients (FRG) were used as inocula to infect day-old turkey poults. The weight gain, jejunal maltase activity, and gross intestinal lesions were used as the parameters of evaluation. Weight gain and maltase activity were reduced (P = 0.001) by the isolated IECs, 0.2 and 0.1 micron filtrates, and FRG when compared with corresponding controls. IEC lysate filtrate (0.1 micron) and FRG were examined under transmission electron microscope (EM). Enveloped, pleomorphic particles varying in size from 60 to 95 nm were observed and termed stunting syndrome agent (SSA). Primary cultures of turkey IECs were used to further isolate and propagate the SSA. Following the fifth passage in the turkey IECs, the cell lysate induced SS in day-old poults. SSA particles were observed under EM after the fifth passage. The results of this study provide evidence that a viral agent has been isolated and identified from IECs of SS-infected poults and is the etiologic agent of SS.

The In Vitro Propagation of Stunting Syndrome Agent

Stunting syndrome (SS) is a viral enteric disease of turkey poults. The etiologic agent (stunting syndrome agent [SSA]) of this disease has been reported recently. The objective of this study was to develop a method for in vitro propagation of SSA. Primary cells, various continuous cell lines, and embryonated eggs were evaluated. Turkey embryos that were inoculated via the amniotic cavity at 24-25 days of incubation were susceptible to SSA infection. The jejunal maltase activity of SSA-inoculated turkey embryos was significantly (P < or = 0.001) lower than that of control embryos. D-xylose absorption was also altered in SSA-infected turkey embryos. The extent of reduction of D-xylose absorption and maltase activity in the infected embryos was nearly identical to that observed when day-old poults were infected with SSA. The intestines from the infected turkey embryos were pale, thin walled, and distended with fluid. Electron microscopic examination of the intestinal fluid and epithelial cell lysate of infected embryos revealed pleomorphic membraned SSA viral particles. SSA that had been serially passaged in turkey embryos retained its ability to induce SS in day-old poults. All the primary and continuous cells that were evaluated did not support the replication of SSA on the basis of cytopathic effects, electron microscopy, and turkey embryo inoculation. Inoculation of chicken embryos by various routes failed to support SSA. All routes of inoculation, other than the amniotic route at 24-25 days, failed to support SSA in turkey embryos. The results of the this study indicate that the SSA was successfully propagated in turkey embryos that exhibited alterations in embryo intestinal absorption and digestive enzyme activity similar to poults with SS. Successful propagation of SSA in turkey embryos should prove beneficial for future studies including characterization of SSA, prevention and control strategies, and enteric disease modeling.

Environmental Impacts of Emergency Livestock Mortality Composting—Leachate Release and Soil Contamination

A 3-year study was conducted in Iowa to evaluate the feasibility of using composting for emergency disposal of cattle mortalities. During the study, 49 metric tons of 450 kg cattle carcasses were composted in 27 replicated unturned windrow test units constructed during three different seasons of the year. Each test unit contained 1.8 metric tons of carcasses enveloped in one of 5 different materials: corn silage, ground cornstalks, straw/manure, leaves, or a soil/compost blend. Due to their water absorbing capacity and ability to evaporate absorbed water, the volume of leachate released into the soil was generally less than 5% of the 500-600 mm of precipitation that fell on the test units. Chemical analysis of 1.2 m deep soil cores collected from beneath the composting test units prior to and following composting showed statistically significant increases in chloride concentrations at all depths beneath composting test units constructed from silage, cornstalks, straw, and the soil/compost blend. Statistically significant increases in % total carbon (silage test units only) and % total nitrogen (silage, cornstalk, straw/manure test units) were limited to the top 15 cm of soil. Increases in these pollutants were moderate, amounting to less than 5X, 0.2X and 0.4 X respectively of chloride, % total carbon, % total N concentrations prior to composting. Statistically significant increases in total ammonia-nitrogen were noted at depths of up to 90 cm beneath test units constructed with silage or leaves, and at 30 cm and 15 cm depths respectively beneath test units constructed with straw/manure and cornstalks. The ammonia-nitrogen increases were large, ranging from 40-160 X of pre-composting levels of ammonia in the topsoil. When compared with the groundwater pollution potential of carcass burial, however, the estimated total mass of N contained in the composted cattle carcasses was 4-10 X the increases in total N measured in the soil beneath the composting test units.

The Sensitivity and Specificity of a Reverse Transcription-Polymerase Chain Reaction Assay for the Avian Pneumovirus (Colorado Strain)

A reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of avian pneumovirus (APV), Colorado strain (US/CO), was evaluated for sensitivity and specificity. The single-tube RT-PCR assay utilized primers developed from the matrix (M) gene sequence of the US/CO APV. The RT-PCR amplified the US/CO APV but did not amplify other pneumoviruses, including the avian pneumoviruses subgroups A and B. The RT-PCR was capable of detecting between 10(0.25) mean tissue culture infective dose (TCID50) and 10(-0.44) TCID50 of the US/CO APV. These results have demonstrated that the single-tube RT-PCR assay is a specific and sensitive assay for the detection of US/CO APV.

Primary cell culture of turkey intestinal epithelial cells.

Primary cell culture has been widely used in various types of studies and proven useful for the isolation and identification of avian pathogens. Difficulties in growing intestinal epithelial cells in vitro have limited their use for such studies. In the present study, a co-culture system was developed for the primary culture of intestinal epithelial cells. A monolayer obtained from 14-to- 16-day-old turkey embryo intestinal fibroblasts was used as a feeder layer. Feeder layers from turkey embryo fibroblasts and from a continuous cell line (mouse 3T3 fibroblasts) were also employed but were not as successful. The intestinal epithelial cells were isolated by dissociation from the intestinal tracts of 1-day-old turkey poults and grown on the feeder layers. Growth and maintenance media were supplemented with various components, including fetal calf serum, chicken serum, hormones, and other growth factors. The epithelial cells grown on feeder layers from the intestinal fibroblasts allowed the intestinal epithelial cells to be maintained in vitro for periods of 7 to 10 days. This technique may prove useful for various applications, including isolation of enteropathogens, and for basic studies of the intestinal tract concerning such subjects as physiology, immunology, and toxicology.

Effect of envelope material on biosecurity during emergency bovine mortality composting.

The biosecurity of composting as an emergency disposal method for cattle mortalities caused by disease was evaluated by conducting full-scale field trials begun during three different seasons and using three different envelope materials. Process biosecurity was significantly affected by the envelope material used to construct the composting matrix. Internal temperatures met USEPA Class A time/temperature criteria for pathogen reduction in 89%, 67%, and 22%, respectively of seasonal test units constructed with corn silage, straw/manure, or ground cornstalks. In trials begun in the winter, survival times of vaccine strains of avian encephalomyelitis and Newcastle disease virus were noticeably shorter in silage test units than in the other two materials, but during summer/spring trials survival times in ground cornstalk and straw/manure test units were similar to those in test units constructed with silage.

Development of a novel method of lytic phage delivery by use of a bacteriophage P22 site‐specific recombination system

Bacteriophage therapy represents a potential alternative to the use of antibiotics to control proliferation of pathogenic bacteria. As an alternative to the strategy where a limited number of doses of large numbers of lytic bacteriophages are administered, a novel method delivery system was developed so that phages are continually released into the culture. Specifically, a non-pathogenic Escherichia coli strain was constructed that was lysogenic for a lytic mutant of bacteriophage lambda. This lysogen was shown to be effective at decreasing the number of lambda-sensitive E. coli in vitro. Construction of this E. coli strain was accomplished by development of a plasmid-based system utilizing the site-specific recombination machinery of bacteriophage P22 to integrate DNA constructs into the host chromosome. This recombination system is useful for strain construction and other genetic manipulations in both E. coli and Salmonella enterica serovars.