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.     

A small round virus associated with enteritis in turkey poults

In a natural outbreak of enteric disease in turkey poults, Salmonella, group D rotavirus, astrovirus, and a small (18-24 nm) round virus were detected in the gut contents. Except for the small virus, the pathogenic potential of the other agents is recognized. In experiments, the small round virus was shown to be transmissible and pathogenic in specific-pathogen-free turkey poults.     

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.     

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.     

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.     

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.     

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.     

Induction of functional defects in macrophages by a poult enteritis and mortality syndrome-associated turkey astrovirus.

The interaction of a poult enteritis and mortality syndrome (PEMS)-turkey astrovirus-Ohio State University (TAst-OSU) with the mononuclear phagocytic system cells, namely macrophages, was examined after in vitro and in vivo exposure. In vitro exposures were performed by incubating adherent turkey macrophages with various volumes of 10(6) 50% embryo infective dose (EID50)/ml TAst-OSU stock, whereas for in vivo challenge, poults were given a 200 microl inoculum of 10(6) EID50/ml TAst-OSU stock at 7 days of age. Results show that TAst-OSU in vitro exposure reduced macrophage viability relative to controls (P < 0.05) and decreased phagocytosis (P < 0.05) and intracytoplasmic killing of Escherichia coli (P < 0.05) after a 42-48-hr exposure. Poults challenged with TAst-OSU in vivo recruited almost 50% fewer Sephadex-elicited inflammatory cells in the abdominal cavity (P < 0.05) as compared with the sham controls. Similar to in vitro exposure, macrophages isolated from in vivo TAst-OSU-challenged poults exhibited reduced percentage of phagocytic macrophages (P < 0.05) as well as fewer intracytoplasmic E. coli per phagocytic macrophage (P < 0.05). TAst-OSU-challenged poults had a greater number of viable E. coli in their spleens (P < 0.05) after an intravenous E. coli challenge as compared with the non-TAst-OSU-challenged control poults. Macrophage-mediated cytokines and metabolites were also examined during this study. Both in vitro and in vivo TAst-OSU challenge resulted in reduced interleukin (IL)-1 and IL-6 activity. On the contrary, nitrite levels in macrophage culture supernatant fraction of TAst-OSU-challenged macrophages were significantly higher (P < or = 0.05). The findings of these studies indicated that TAst-OSU challenge induced defects in macrophage effector functions, implying that PEMS-turkey astrovirus can potentially impair the immune response of turkeys, thereby leading to enhanced susceptibility of turkeys to secondary, perhaps even fatal, bacterial infections.     

Skeletal lesions associated with a naturally occurring poult enteritis

One-day-old poults were placed on contaminated litter on which poults previously had developed an enteric disease characterized by diarrhea, increased mortality, and stunting. These exposed birds were examined for clinical signs and pathologic changes in bone and parathyroid glands compared with controls. Intestinal and fecal samples were examined for potential pathogens. Exposed poults varied in size as early as day 8 and had significantly decreased weight gains and reduced shank lengths on days 8, 12, 16, and 21. The proximal tibial growth plate was narrowed. The mineralized hypertrophy zone was decreased in length and contained multiple non-mineralized bands on days 8, 12, 16, and 21. Metaphyseal trabeculae were reduced in amount on days 16 and 21. Parathyroid glands were hyperplastic on days 16 and 21. The bone and parathyroid gland lesions indicated that mineral homeostasis was being maintained at the expense of the skeleton during the enteric disease. A specific etiology for the enteric disease was not determined. Cryptosporidium, rotavirus, paramyxovirus, and Salmonella were identified in the exposed poults, and paramyxovirus and Salmonella were identified in the controls.     

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.     

Enteric disease in specific-pathogen-free turkey poults inoculated with a small round turkey-origin enteric virus

Four- and 5-day-old specific-pathogen-free turkey poults were inoculated orally or by contact exposure to a small round turkey-origin enteric virus. At days 4 and 8 postinoculation (PI), the orally inoculated poults had significantly lower body weight gains than control poults. Poults at day 4 (orally inoculated) and 5 (contact-exposed) PI had watery droppings, dilated thin-walled ceca filled with yellow foamy fluid, catarrhal small intestinal secretions, pale intestinal serosa, and mild lymphocytic enteritis. In addition, at day 4 PI, poults were lymphopenic, had intracytoplasmic crystalline arrays of 17.1 +/- 1.1 nm viral particles in the jejunal villar enterocytes, and had an 18-to-24-nm virus in intestinal contents. Analysis of morphometric data revealed mild shortening of villi in the duodenum and elongation of crypts in the duodenum and ileum during the late stage of the syndrome (day 8 PI). These findings suggest that the 18-to-24-nm virus can produce an enteric disease syndrome and that the acute clinical manifestation of this syndrome is not the result of morphologic change such as intestinal villus atrophy. The definitive identity of this 18-to-24-nm virus is not known; however, based on size and intracytoplasmic arrays of virus, it is most probably an enterovirus.     

Viral agents associated with outbreaks of diarrhea in turkey flocks in Quebec.

The relative importance of various enteric viruses associated with diarrhea of turkey poults was investigated by an evaluation of specimens received since 1982. Specimens originated from one to eight week old turkey poults, with mild to severe diarrhea, from 114 flocks in 42 commercial operations located in southern Quebec. The acute phase of enteritis occurred usually in poults between two and four weeks of age. Clarified intestinal contents were examined by direct electron microscopy and enzyme immunoassays. Enzyme-linked immunosorbent assays were performed with antisera to bovine rotavirus group antigen, avian reovirus types 1 to 5, and the prototype strain of the turkey enteric coronavirus. The presence of viruses could be demonstrated by electron microscopy in 55.3% of the specimens, and at least five different viruses were incriminated either alone or in combination. The coronavirus was by far the most common enteric virus with a prevalence of 47.5%. By enzyme-linked immunosorbent assay, rotavirus, reovirus and turkey coronavirus were detected in 14.5%, 18.1% and 61.4% of the specimens, respectively. By electron microscopy, 56.6% of these cases were positive for at least one virus.     

Electron microscopic identification of viruses associated with poult enteritis in turkeys grown in California 1993-2003

Poult enteritis (PE) is one of the most common diseases seen in young turkey flocks. Since 1993, more than 1800 cases of suspected PE have been submitted for examination by negative stain electron microscopy; this has involved more than 2400 individual results, because in many cases more than one virus was identified; at least 1500 individual results were positive for viruses. Viruses have been identified in poults as young as 3 days and up to 9 wk of age. The most commonly found viruses are rotavirus-like viruses and small round viruses ranging from 15 nm to 30 nm, either alone or in combination. Reovirus, birnavirus, and adenovirus have also been detected. There has been no evidence to suggest the presence of coronaviruses. This report summarizes our findings.     

Propagation of virulent and avirulent turkey hemorrhagic enteritis virus in cell culture

Virulent and apathogenic isolates of turkey hemorrhagic enteritis virus (HEV) were successfully propagated in lymphoblastoid cell lines of turkey origin, whereas spleen and kidney cell cultures from HEV-infected turkeys failed to replicate the virus. The lymphoblastoid cell lines used were MDTC-RP16 and MDTC-RP19, which were previously established from tumors induced by Marek's disease virus in turkeys. Virus replication followed co-cultivation of lymphoblastoid cells with spleen cells from HEV-infected turkeys. Virus replication was demonstrated by immunofluorescence, by agar-gel-precipitin tests, and by electron microscopy. Supernatant fluid of cultures infected with virulent HEV caused death and specific lesions in turkey poults. Poults vaccinated with apathogenic HEV were protected against death and lesions after challenge with pathogenic HEV, which was recovered from infected cultures. The MDTC-RP19 cell line appeared far more susceptible than the MDTC-RP16 cell line to infection with HEV.     

Multifocal eosinophilic enteritis associated with a small intestinal obstruction in a standardbred horse

A seven-year-old standardbred gelding developed marked signs of colic associated with an acute small intestinal obstruction. Surgical exploration revealed three intramural, circumferential constricting lesions in the small intestine, the two most severe of which were in the jejunum and were resected. The horse was euthanased owing to postoperative complications. Histopathological examination confirmed the diagnosis of idiopathic multifocal eosinophilic enteritis.