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.     

Risk factors associated with poult enteritis mortality syndrome-positive turkey flocks

Poult enteritis mortality syndrome (PEMS) has been an economically devastating disease in North Carolina since the early 1990s. Though much is known about the disease, many questions remain unanswered about the syndrome, including its cause, transmission of causative agent(s), and control methods. This study was designed to investigate the association between PEMS and farm management factors. A prospective longitudinal study was conducted by collecting farm data and monitoring weekly mortality in 54 commercial turkey flocks raised in PEMS-affected regions. Univariate and multivariate statistical analyses revealed that enhancing rodent control methods was negatively associated (P = 0.0228) with PEMS.     

Isolation of a reovirus from poult enteritis and mortality syndrome and its pathogenicity in turkey poults

Poult enteritis and mortality syndrome (PEMS) is an acute, infectious intestinal disease of turkey poults, characterized by high mortality and 100% morbidity, that decimated the turkey industry in the mid-1990s. The etiology of PEMS is not completely understood. This report describes the testing of various filtrates of fecal material from control and PEMS-affected poults by oral inoculation into poults under experimental conditions, the subsequent isolation of a reovirus, ARV-CU98, from one of the PEMS fecal filtrates, and in vivo and in vitro studies conducted to determine the pathogenicity of ARV-CU98 in turkey poults. In order to identify a filtrate fraction of fecal material containing a putative etiologic agent, poults were challenged in two independent experiments with 220- and 100-nm filtrates of fecal material from PEMS-negative and PEMS-positive poults. The 100-nm filtrate was chosen for further evaluation because poults inoculated with this filtrate exhibited mortality and significantly lower (P < or = 0.05) body weight and relative bursa weight, three clinical signs associated with PEMS. These results were confirmed in a third experiment with 100-nm fecal filtrates from a separate batch of PEMS fecal material. In Experiment 3, body weight and relative bursa and thymus weights were significantly lower (P < or = 0.05) in poults inoculated with 100-nm filtrate of PEMS fecal material as compared with poults inoculated with 100-nm filtrate of control fecal material. Subsequently, a virus was isolated from the 100-nm PEMS fecal filtrate and propagated in liver cells. This virus was identified as a reovirus on the basis of cross-reaction with antisera against avian reovirus (FDO strain) as well as by electrophoretic analysis and was designated ARV-CU98. When inoculated orally into poults reared under controlled environmental conditions in isolators, ARV-CU98 was associated with a higher incidence of thymic hemorrhaging and gaseous intestines. In addition, relative bursa and liver weights were significantly lower (P < or = 0.05) in virus-inoculated poults as compared with controls. Virus was successfully reisolated from virus-challenged poults but not from control birds. Furthermore, viral antigen was detected by immunofluorescence in liver sections from virus-challenged poults at 3 and 6 days postinfection and virus was isolated from liver at 6 days postinfection, suggesting that ARV-CU98 replicates in the liver. In addition to a decrease in liver weight, there was a functional degeneration as indicated by altered plasma alanine aminotransferase and aspartate aminotransferase activities in virus poults as compared with controls. Although this reovirus does not induce fulminating PEMS, our results demonstrated that ARV-CU98 does cause some of the clinical signs in PEMS, including intestinal alterations and significantly lower relative bursa and liver weights. ARV-CU98 may contribute directly to PEMS by affecting the intestine, bursa, and liver and may contribute indirectly by increasing susceptibility to opportunistic pathogens that facilitate development of clinical PEMS.     

Mortality patterns associated with poult enteritis mortality syndrome (PEMS) and coronaviral enteritis in turkey flocks raised in PEMS-affected regions.

Poult enteritis mortality syndrome (PEMS) is an economically devastating disease. To date, many questions about the syndrome remain unanswered, including its cause, transmission of causative agent(s), and control methods. Turkey coronavirus (TCV) infection has been associated with some outbreaks of PEMS, with areas having a higher prevalence of TCV infection also experiencing an increased incidence of PEMS. This study was designed to establish mortality patterns for flocks experiencing excess mortality and TCV infection in PEMS-affected regions and to delineate the possible role of TCV in PEMS-affected flocks. Fifty-four commercial turkey flocks on farms in areas with and without a history of TCV infection were monitored for weekly mortality and for antibodies to TCV. Flocks were chosen on the basis of placement dates and were monitored from day of placement until processing. All flocks were tested for TCV by an indirect fluorescent antibody assay. PEMS status was determined with the use of the clinical definition of mortality greater than 2% during any 3-wk period from 2 wk of age through the end of brooding due to unknown cause. Of the 54 flocks, 24 remained healthy, 23 experienced PEMS, and 7 tested positive for TCV but did not experience PEMS. Ten flocks experienced PEMS and tested positive for TCV, whereas 13 flocks experienced PEMS and did not test positive for TCV. Four health status groups were evident: healthy, PEMS positive, TCV positive, and PEMS + TCV positive. Distinct mortality patterns were seen for each of the four health status groups. Whereas TCV was associated with PEMS in 43% of PEMS cases, 13 cases (57%) of PEMS did not involve TCV. Additionally, 7 out of 17 cases of TCV (41%) did not experience excess mortality (PEMS) at any time during brooding of the flock. The results of this study indicate that TCV can be associated with PEMS but is neither necessary nor sufficient to cause PEMS.     

Viral agents associated with poult enteritis and mortality syndrome: the role of a small round virus and a turkey coronavirus

Intestinal samples from turkey poults affected with poult enteritis and mortality syndrome (PEMS) were examined for viruses by immune electron microscopy and double-stranded RNA virus genome electropherotyping. Turkey coronavirus (TCV), avian rotaviruses, reovirus, and a yet undefined small round virus (SRV) were detected. The SRV and TCV were isolated and propagated in turkey embryos. Challenge of specific-pathogen-free turkey poults with SRV, TCV, or both resulted in mortality and clinical responses similar to those of natural PEMS. Our experiments indicate that SRV and TCV are possibly important agents in the etiology of PEMS and the combination of these infections might result in outbreaks with high mortality. The severity of clinical signs and mortality of PEMS are postulated to be partly related to the virus agents involved in individual outbreaks.     

Alterations in macrophage-produced cytokines and nitrite associated with poult enteritis and mortality syndrome

Poult enteritis and mortality syndrome (PEMS) is an acute, transmissible, infectious intestinal disease associated with high mortality and morbidity in turkey poults. Earlier studies demonstrated immune dysfunction, involving both humoral and cell-mediated immunity, associated with PEMS. The current study examined cytokines and metabolites produced by macrophages from poults exposed to PEMS agent(s). Six trials were conducted with six separate hatches of poults. Poults in the PEMS group were exposed to PEMS agent(s) via contact exposure at 7 days of age whereas uninfected poults served as control poults. Abdominal macrophages were harvested from control (uninfected) and PEMS poults at various times postexposure and cultured for 18-24 hr in the presence of Escherichia coli lipopolysaccharide. Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) bioactivities and nitrite levels in macrophage culture supernatants were quantified. Macrophage supernatants from PEMS poults had greater IL-1-mediated stimulation index compared with the macrophage supernatants from uninfected control poults in both trials. However, this increase was significant only in trial 1. IL-6 activity tested in three separate trials was significantly higher in PEMS macrophage supernatants over the controls. On the contrary, TNF-alpha production by macrophages was decreased in PEMS macrophage culture supernatants. Nitrite levels in PEMS macrophage culture supernatants were significantly higher in two out of three trials. These findings suggest that the enhanced production of proinflammatory cytokine/metabolites by activated macrophages in PEMS poults may be responsible, at least in part, for the physiological intestinal inflammation, gut motility, and anorexia that characterize this disease.     

Poult malabsorption syndrome. I. Malabsorption in poult enteritis.

One-day-old poults were placed on littler on which poults had previously developed diarrhea, increased mortality, and stunting. Small intestines, pancreas, and liver were evaluated histologically. Morphometric evaluations were conducted to determine villous length and crypt depth. Poults were evaluated for malabsorption utilizing D-xylose and lipid absorption tests. Compared with controls, the gastrointestinal tract of affected birds was grossly distended, was fluid-filled, and had thin, flaccid walls on days 5 and 8. Ceca were distended with brown watery fluid and gas on days 5, 8, and 12. No histologic lesions were present in the liver, pancreas, or pancreatic ducts, and only mild inflammatory changes were present in the small intestine. Villous atrophy and crypt hypertrophy were present in the small intestine on days 5, 8, 12, 16, and 21. Morphometry revealed significant decreases in villous lengths and increases in crypt depth throughout the trial. D-Xylose and lipid absorption were significantly decreased on days 8 and 11. Intestinal epithelial damage by infectious agents with subsequent villous atrophy is postulated to have produced malabsorptive diarrhea.     

Characterization of turkey coronavirus from turkey poults with acute enteritis.

The present study was to characterize turkey coronavirus associated with turkey poult enteritis and mortality. Intestinal contents or intestines from affected turkey poults and inoculated turkey embryos contained coronaviruses as revealed by electron microscopy or were positive for turkey coronavirus by immunofluorescent antibody assay. Sucrose density gradient ultracentrifugation of the virus-containing intestinal homogenate yielded two opalescent bands corresponding to the buoyant densities of 1.14-1.15 and 1.18-1.20 g/ml, respectively. Coronaviral particles from intestinal contents or the sucrose density gradient preparation were mainly spherical in shape and had envelope and central depression. They were surrounded by a fringe of regularly spaced petal-shaped projections attached to the particles by a short stalk. Purified viruses hemagglutinated rabbit erythrocytes with a titer of 16. Major protein bands of purified viruses analyzed by SDS-PAGE were located at 200, 100-110, 50-60, and 30-35 kDa. The patterns of protein bands were consistent with those of Minnesota or Quebec turkey coronavirus isolates. A 568 bp nucleotide fragment of turkey coronavirus spike protein gene was amplified from RNA of inoculated turkey embryo intestine or purified virus. Sequence analysis of the 568 bp PCR product revealed high degree of identity with the corresponding spike protein gene sequence of human and bovine coronaviruses. The results indicated that turkey coronavirus was associated with turkey poults with acute enteritis.     

A novel "small round virus" inducing poult enteritis and mortality syndrome and associated immune alterations

The role of a novel "small round virus" (SRV) isolated from poult enteritis and mortality syndrome (PEMS) cases in inducing PEMS and associated immune alterations was examined in this study. Specific-pathogen-free and conventional poults were orally challenged with SRV and/or turkey coronavirus and monitored for clinical signs. Intestines, thymus, bursa, and spleens were examined for SRV antigen at various days postinoculation (DPI). Peripheral blood lymphocytes (PBLs), thymocytes, and splenic lymphocytes from inoculated poults or lymphocytes isolated from healthy poults after incubation with SRV in vitro were examined for lymphoproliferative potential against concanavalin A (Con A). The incidence of lymphocyte subpopulations in the peripheral blood and thymic lymphocytes of SRV-challenged poults was examined by flow cytometry. The results of these studies showed that the SRV challenge induced diarrhea, growth suppression, and atrophy of thymus and bursa resembling those of PEMS in field and/or experimental infections. The SRV antigen was detected in intestinal tissues soon after infection (i.e., at 2 and 4 DPI), whereas lymphoid tissues such as thymus, bursa, and spleen were positive for SRV antigen starting at 4 DPI until 8 DPI, suggesting virus translocation to lymphoid organs. The responsiveness of PBLs to Con A at 2 DPI was significantly reduced in all virus challenge groups (e.g., 28% and 22% in the SRV-alone group in studies 1 and 2, respectively) below the uninfected group. However, this suppressed response was no longer evident in the SRV group by 7 DPI. The SRV incubation with normal thymocytes and splenocytes in vitro resulted in significantly reduced lymphoproliferative response against Con A (41.2% and 10.49% reductions at 1:50 SRV dilution vs. controls in thymocytes and splenocytes, respectively). Flow cytometry analysis revealed a sudden decline at 2 DPI in the numbers of CD4- CD8+ lymphocyte subset in PBLs of SRV-infected poults. However, by 8 DPI, SRV-challenged poults had relatively higher CD4- CD8+ lymphocytes in PBLs. On the contrary, thymocytes had higher percentages of CD4- CD8+ lymphocytes at 2 and 4 DPI and reached comparable levels at 8 DPI in controls and SRV-infected poults. No differences were observed in CD4+ CD8- lymphocyte numbers in controls vs. SRV-infected poults. The findings of these studies imply that SRV may be a promising primary etiologic agent of PEMS. Furthermore, the SRV infection may compromise the lymphocyte-mediated immune defenses by reducing lymphoproliferation and the CD4- CD8+ (presumably T-cytotoxic cells) lymphocytes during the acute stage of SRV infection.     

Identifying agent(s) associated with poult enteritis mortality syndrome: importance of the thymus

Poult enteritis mortality syndrome (PEMS), a highly infectious disease of young turkeys, causes serious financial losses to the turkey industry. Clinically, PEMS is defined by mortality profiles, diarrhea, growth depression, and immunosuppression. Although many viruses, bacteria, and parasites are found in PEMS-infected birds, the inciting agent remains unknown. Experimentally, PEMS can be reproduced by exposing na?ve poults to the intestinal contents from infected birds. Previous reports suggest that extraintestinal tissues fail to reproduce the disease. Histopathologic examination of tissues from PEMS-infected poults suggested that the thymus exhibited the earliest signs of pathology. On the basis of these observations, we hypothesized that the thymus harbors an agent(s) involved in PEMS. In these studies, na?ve turkey poults were orally inoculated with a bacteria-free filtrate composed of either the intestines and feces or the thymus from PEMS-infected birds and were monitored for clinical signs of PEMS. Poults exposed to a filtrate composed solely of the thymus from PEMS-infected birds exhibited diarrhea, growth depression, mortality, pathology, and, most importantly, immunosuppression similar to poults exposed to the intestinal filtrate. The results of this study suggest that the thymus of infected birds harbors the agent(s) that can reproduce a PEMS-like disease in turkey poults.     

Hemorrhagic enteritis virus inclusions in turkey renal tubular epithelium.

Commercial turkeys from four Iowa flocks, two Illinois flocks, and three California flocks were submitted to state diagnostic laboratories because of a variety of health problems. The turkeys ranged in age from 5 to 12 weeks, included both hens and toms, and were owned by five different companies. Some flocks had previously been immunized with live hemorrhagic enteritis vaccine, and other flocks were unvaccinated. In all accessions, basophilic intranuclear inclusion bodies were observed in renal tubular epithelium by light microscopy. Transmission electron microscopy showed that the inclusions consisted of densely packed virus particles. The virions were identified as adenoviruses based upon the icosahedral morphology and average particle diameters of 72 nm. Avidin-biotin immunoperoxidase staining of formalin-fixed, paraffin-embedded kidneys was used to identify this adenovirus as hemorrhagic enteritis virus.     

Induction, collection, and partial characterization of induced respiratory macrophages of the turkey

Respiratory macrophages (RM) of the turkey were elicited with a 1:4 (v/v) suspension of incomplete Freund's adjuvant in sterile phosphate-buffered saline injected directly into the abdominal air sacs. RM were purified by passage through a Ficoll-Hypaque gradient resulting in 95.7 +/- 5.9% purity and 94.8 +/- 12.3% viability. On days 7 and 9 postinjection, adequate numbers (7.15 +/- 5.47 X 10(6) macrophages per turkey) of RM for in vitro experiments were obtained. RM of the turkey demonstrated the ability to adhere to glass, phagocytize Zymosan A, and kill Escherichia coli in vitro.     

Investigation of field outbreaks of turkey haemorrhagic enteritis in Hungary

Epidemiological, pathological, serological and virological investigations are reported on turkey haemorrhagic enteritis virus (THEV) infection in Hungarian turkey flocks. The pathogenesis of infection in experimentally infected turkeys and chickens, as well as the usefulness of polymerase chain reaction (PCR)/sequencing method for epidemiological investigation and for the differentiation of vaccine and field strains of THEV was also studied. Since the first recognition of the disease in Hungary in the late 1970s, until recently the disease has been diagnosed sporadically in its mild form. In the last few years (2000-2005), however, the number of outbreaks and the severity of the disease increased (9-23 affected flocks/year). Most of the outbreaks occurred at the age of 6 to 8 weeks and was complicated with Escherichia coli infection. The antibody levels to THEV in turkey flocks gradually declined till 5-7 weeks of age, and then they increased sharply due to natural infection with THEV. The immune response to vaccination (at 5 weeks of age) showed no significant antibody level increase one week postvaccination, but four weeks later the antibody level reached high values and then remained at this high level. The agar gel immunodiffusion (AGID) test to detect turkey adenovirus A (TAdV-A) antigen and PCR methods for THEV-specific DNA gave similarly positive results if spleens with pathognomonic lesions were tested; however, PCR proved to be more sensitive in cases with less characteristic pathological lesions. Nucleotide sequence alignment of PCR products amplified from Hungarian field strains and the Domermuth vaccine strain and that of the published THEV hexon sequences in GenBank database revealed slight differences between the sequences.