Hemorrhagic enteritis of turkeys: influence of maternal antibody and age at exposure

The effect of maternal antibody (MAB) to hemorrhagic enteritis (HE) on the response of turkeys to infection with virulent and avirulent strains of HE virus (HEV) was examined. The influence of age at exposure and treatment with HEV antibody on development of clinical HE also was studied. MAB protected poults from clinical HE for up to 6 weeks of age. MAB also interfered with vaccination against the disease for at least 5 weeks after hatching, as indicated by absence of HEV antigen in spleens and by poor seroconversion at 6 days and at 3 weeks post-vaccination, respectively. The incidence of clinical HE in MAB-negative poults was significantly higher in poults inoculated with virus at 15 days of age or older than in poults inoculated at 1-13 days of age. Further, MAB-negative poults embryonally inoculated with virulent or avirulent strains of HEV did not develop disease; these poults developed antibody and resisted challenge with virulent virus at 6 weeks of age. Poults treated with HE antibody within 1 hour of challenge or at 1, 3, or 5 weeks before challenge with virulent virus were protected against lesions and mortality induced by HEV. These results suggest that MAB may influence susceptibility of turkeys to infection with HEV for at least 5 to 6 weeks after hatching, unlike the case with most other viral infections of poultry. The results confirm that early age resistance to clinical HE is independent of MAB and suggest that such resistance persists for up to 13 days of age. The data also suggest that turkeys lacking MAB can be immunized against HE by embryo vaccination.     

Role of splenectomy in prevention of hemorrhagic enteritis and death from hemorrhagic enteritis virus in turkeys

Hemorrhagic diarrhea, gross hemorrhagic enteritis, and death caused by intravenous virus injection of hemorrhagic enteritis virus (HEV) were prevented in otherwise susceptible turkey poults by surgical splenectomy. The splenectomized poults produced anti-HEV antibodies, which indicated that splenectomy did not completely prevent replication of the virus. These results indicate that the spleen is necessary for the development of the intestinal lesions of this disease. The role of a toxic factor in this disease is discussed.     

Efficacy and safety of a cell-culture live virus vaccine for hemorrhagic enteritis of turkeys: laboratory studies

Poults free from hemorrhagic enteritis (HE) antibody were vaccinated by gavage at 1 day or 2 weeks of age with a live HE vaccine virus that had been propagated in a Marek's disease (MD)-induced B-lymphoblastoid cell line of turkey origin. Vaccinated and unvaccinated poults were challenged with a virulent HE virus at various times postvaccination. One hundred tissue-culture-infectious doses of the vaccine virus per poult were sufficient to induce a serological response as well as to protect poults against HE lesions and mortality. Vaccinated poults were protected against the disease as early as 1 week and as late as 8 weeks PV. The vaccine was efficacious by several routes of application. The vaccine virus spread horizontally from vaccinated to contact-exposed poults, as indicated by seroconversion and resistance of contact-exposed poults to challenge. The vaccine had no detectable harmful effects on the humoral immune response to particulate antigens or on weight gain of vaccinated poults. The vaccine proved to be free from MD virus, as indicated by the absence of MD lesions and antibody in 8-week-old chickens inoculated intra-abdominally with the vaccine at hatching. These findings indicate that the cell-culture-propagated HE vaccine is efficacious and safe.     

Field studies with convalescent serum and infectious bursal disease vaccine to control turkey coryza

Convalescent serum given to 1-day-old poults delayed clinical signs of turkey coryza by several days and reduced mortality on infected farms. Turkey breeders immunized with cell-culture-adapted infectious bursal disease virus (IBDV) or turkey infectious bursal disease virus (TIBDV) had a marked increase in virus-neutralization (VN) antibody titers. The VN antibody titer was significantly higher in progeny poults than in poults from unimmunized breeders. Clinical turkey coryza and mortality was considerably less in poults from IBD- or TIBD-vaccinated breeders than in control poults. They also responded more favorably to hemorrhagic enteritis and fowl cholera vaccination.     

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.     

Preliminary testing in turkeys of the safety and efficacy of a putative haemorrhagic enteritis virus vaccine

Haemorrhagic enteritis virus (HEV) causes clinical haemorrhagic enteritis in young poults and/or subclinical immunosuppression which is often associated with colibacillosis. This disease is controlled with live vaccines worldwide, however, importation of HEV vaccines or cells that support HEV propagation are not permitted in Australia. A major experiment in isolators was conducted to test the safety and efficacy of a putative HEV vaccine. The study had a factorial design with four factors namely vaccination age (28 and 42 days of age), vaccine dose (0, 10⁵, 10⁶, 10⁷ genomic copies of HEV vaccine), challenge with HEV (yes, no) and vaccination‐challenge interval (7, 21 or 42 days). A total of 315 poults were used providing 6‐8 birds per treatment combination. Turkey growth rate, mortality, pathological findings, anti‐HEV antibodies and viral load were examined. Vaccination lead to significant increases in anti HEV antibody over the following 2‐4 weeks. Overall, vaccination with 10⁶ and 10⁷ was protective against increase in relative splenic weight and splenic viral load in challenged birds. Clinical haemorrhagic enteritis was not induced by any treatment but there was an increased incidence of airsacculitis in groups receiving either HEV vaccine or challenge virus compared to the negative control birds (25.8‐29.3% vs. 9.4%, P < 0.05). Growth rate, mortality and relative bursal weight were unaffected by vaccination. This laboratory level study indicates that the putative vaccine is safe and likely to be efficacious, but may cause elevated levels of airsacculitis. These findings require confirmation in larger scale field trials.     

An enzyme-linked immunosorbent assay for detection of Bordetella avium infection in turkey flocks: sensitivity, specificity, and reproducibility.

An enzyme-linked immunosorbent assay (ELISA) for detection of Bordetella avium infection in turkey poults was developed. One-week-old poults challenged intratracheally with 10(12) colony-forming units of B. avium had detectable titers (greater than or equal to 11), with an average of 13.6% positive samples when the birds were 6 to 11 weeks old. The method was sensitive enough to detect maternal antibodies to B. avium in poults up to 3 weeks of age. The same poults challenged at 1 week of age had 100% tracheal infection up to 3 weeks of age, which dropped to 0% by 6 weeks. The method resulted in no false-positive samples (titer = 0) from birds not infected with B. avium and tested weekly between 4 and 11 weeks of age. Antibodies in turkey flocks infected with Newcastle disease virus, hemorrhagic enteritis virus, and Mycoplasma meleagridis, and birds infected with Escherichia coli had no apparent cross-reactivity to the B. avium antigens used in the ELISA. The percentages of B. avium-positive serum samples collected from different turkey flocks did not significantly differ (P greater than 0.05) when samples were tested by the developed ELISA at different times, an indication of the reproducibility of the method.     

Colibacillosis of turkeys exacerbated by hemorrhagic enteritis virus. Laboratory studies

Lesions typical of colibacillosis disease were reproduced in laboratory experiments. Mortality resulting from experimentally produced colibacillosis was significantly increased when Escherichia coli O1:K1 was presented to poults that had been orally inoculated with hemorrhagic enteritis virus (HEV) 1 week earlier. These and previous data suggest that HEV infection can exacerbate colibacillosis of older poults. HEV infection apparently damages the poults' defense system enough to account for the observed increase in susceptibility to E. coli.     

Pathology and virus tissue distribution of Turkey origin reoviruses in experimentally infected Turkey poults

The pathogenesis of 4 isolates of turkey-origin reovirus (NC/SEP-R44/03, NC/98, TX/98, and NC/85) and 1 chicken-origin reovirus (1733) was examined by infecting specific pathogen free (SPF) poults. These turkey-origin reovirus (TRV) isolates were collected from turkey flocks experiencing poult enteritis and are genetically distinct from previously reported avian reoviruses. Microscopic examination of the tissues collected from the TRV-infected poults revealed different degrees of bursal atrophy characterized by lymphoid depletion and increased fibroplasia between the bursal follicles. To understand the relationship between virus spread and replication, and the induction of lesions, immunohistochemical staining (IHC) for viral antigen, in situ hybridization (ISH) for the detection of viral RNA, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for the detection of apoptosis in affected tissues was performed. Both IHC and ISH revealed viral antigen and RNA in the surface epithelial cells of the bursa, in macrophages in the interstitium of the bursa and, to lesser degree, in splenic red pulp macrophages and intestinal epithelial cells. Increased apoptosis of bursal lymphocytes and macrophages was observed at 2 and 5 days postinoculation. No lesions were found in tissues from poults inoculated with the virulent chicken-origin strain, however viral antigen was detected in the bursa and the intestine. Although all TRVs studied displayed similar tissue tropism, there were substantial differences in the severity of the lesions produced. Poults inoculated with NC/SEP-R44/03 or NC/98 had moderate to severe bursal atrophy, whereas poults inoculated with TX/98 or NC/85 presented a mild to moderate bursal lymphoid depletion. The lymphoid depletion observed in the bursa appears to be the effect of an indirectly induced apoptosis and would most likely result in immune dysfunction in poults infected with TRV.     

Further studies on in vitro and in vivo assays of hemorrhagic enteritis virus (HEV)

Isolation of hemorrhagic enteritis virus (HEV) from spleens of infected turkeys in the MDTC-RP19 lymphoblastoid cell line was compared with detection of HEV antigen in the same spleens using the agar gel precipitation (AGP) test. A concordance of 80% was found between the two assays. Virus isolation had a sensitivity of 84% and a specificity of 88% compared with the AGP test. RP19 cells were also susceptible to infection with several other avian adenoviruses, but such infection was easily differentiated from that of HEV by a fluorescent-antibody (FA) test. Turkeys required 10(2) tissue-culture-infectious doses (TCID) to develop HE-specific lesions and 10(5) TCID to be killed. On the other hand, as little as 10 TCID of apathogenic HEV protected the poults against challenge with virulent HEV. The enzyme-linked immunosorbent assay (ELISA) for detection of HEV antibody was improved by using virus-infected RP19 cells as antigen. The ELISA appears to be more sensitive than the serum-neutralization test.     

Quantitation of hemorrhagic enteritis virus antigen and antibody using enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays (ELISAs) were developed to quantitate hemorrhagic enteritis virus (HEV) antibodies in turkey sera and HEV antigens in tissue extracts. These assays were more sensitive than the commonly used agar-gel precipitin tests in detecting antigen and antibody. The antibody-ELISA was used to monitor the presence and decline of passive antibodies in turkey poults and the seroconversion of turkeys infected with HEV. The antigen-ELISA was carried out using a monoclonal antibody; this test was used to quantitate HEV antigen in experimentally infected turkeys in a time-sequence experiment. Both ELISAs measured a strong antigenic relationship between an avirulent strain (HEV-A) and a virulent strain (HEV-V).     

Hemorrhagic enteritis: virus distribution and sequential development of antibody in turkeys

Turkeys poults were inoculated intraperitoneally with hemorrhagic enteritis virus (HEV) at 4-1/2 weeks of age. Antibody response and sequential development of viral antigen in various tissues were monitored. An enzyme-linked immunosorbent assay (ELISA) was developed to study antibody production, and immunoperoxidase staining was used to determined sites of localization of the viral antigens in tissues. Results of ELISA and immunodiffusion tests were compared. ELISA detected antibody from day 3 post-infection (p.i.), and gel diffusion detected antibody from day 5 p.i. Peak ELISA antibody titer appeared from day 14 p.i. HEV antigen was detected from 2-6 days p.i. in the spleen, liver, intestine, kidney, and bone marrow; peak titers in the spleen were on day 3 p.i. Virus was not detected after day 6 p.i.     

Relationship between age of flock seroconversion to hemorrhagic enteritis virus and appearance of adenoviral inclusions in the spleen and renal tubule epithelia of turkeys.

A retrospective study was conducted to evaluate the temporal relationship between flock seroconversion to hemorrhagic enteritis virus (HEV) and the appearance of adenoviral inclusions in the spleen and renal tubular epithelium. The study was conducted on samples of turkey poults submitted to the Fresno Branch of the California Veterinary Diagnostic Laboratory System during May to December 1988. The study included 78 submissions (four to eight poults per submission) of ages ranging from 6 to 15 weeks. Sera were tested for antibodies to HEV using the agar gel immunodiffusion test. Spleen and kidney samples were examined by light microscopy for the presence of inclusions in the mononuclear phagocytes of the spleen or in the renal tubular epithelium of the kidney. Logistic regression statistical analysis was used to evaluate the association between the age of the bird and the likelihood of the presence of inclusions in the spleen and kidney, as well as the likelihood of seroconversion to HEV. A significant association (P less than 0.05) was found between the presence of splenic inclusion bodies and the age of the bird. The probability of splenic inclusions was higher in younger birds (6 weeks of age), and decreased as the birds became older, approaching zero at 11 weeks of age. The kidney inclusions were significantly associated with age. The probability of detecting the inclusions increased with age, reached a maximum at 10 weeks, and then declined, approaching zero by 14 weeks. However, the probability of seroconversion to HEV increased significantly with age up to 10 weeks and then remained positive throughout the remainder of the study period.     

Field vaccination against hemorrhagic enteritis of turkeys by a cell-culture live-virus vaccine

A cell-culture-propagated (CC) live-virus hemorrhagic enteritis (HE) vaccine was evaluated for efficacy and safety in two field trials conducted in North Carolina (NC) and Minnesota (MN). At 4 or 5 1/2 weeks of age, 9,839 poults in NC and 15,857 poults in MN were vaccinated with a CC HE vaccine administered via the drinking water. A comparable number of poults were maintained as unvaccinated controls. Vaccinated and unvaccinated poults were compared for seroconversion, response to laboratory challenge with a virulent HE virus at 3 weeks postvaccination, livability, percentage graded A, and average weight at marketing. In both trials, vaccination with the CC HE vaccine resulted in immunity against HE as indicated by seroconversion and by resistance to HE lesions following laboratory challenge with virulent HE virus. Compared with unvaccinated groups, vaccinated groups had a significantly higher percentage of turkeys graded A in the NC trial and in two of three flocks in the MN trial (P less than 0.005). Further, in the NC trial, livability was significantly higher (P less than 0.005) in vaccinated turkeys than in unvaccinated turkeys. These data indicate that the CC HE vaccine is efficacious and safe to use in the field.     

Some observations on the response of ring-necked pheasants to inoculation with various strains of cell-culture-propagated type II avian adenovirus

The response of ring-necked pheasants to inoculation with three strains of cell-culture-propagated type II avian adenovirus was examined. Marble spleen disease (MSD) virus of pheasants and both avirulent and virulent strains of hemorrhagic enteritis virus (HEV) of turkeys all induced typical gross and microscopic splenic lesions of MSD; neither MSD-associated lung lesions nor mortality were noted in inoculated pheasants, regardless of strain of virus used. Pheasants inoculated with a cell-culture-propagated avirulent strain of HEV were properly immunized against challenge with virulent HEV, as indicated by seroconversion and by protection against virus-induced splenic lesions. We conclude that these strains of type II avian adenovirus are comparable in pathogenicity for pheasants and cannot be distinguished. Further, absence of MSD-associated lung lesions and mortality in pheasants maintained under controlled laboratory conditions suggest that other environmental factors are probably involved in induction of such lesions and mortality in field cases of MSD.     

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.     

Field outbreaks of colibacillosis of turkeys associated with hemorrhagic enteritis virus

In a study of field material and a survey conducted by the authors, typical signs of colibacillosis of 6-to-12-week-old poults included sudden onset, listlessness, rales, and high mortality. Signs persisted for about 2 weeks and were often followed by a low incidence of lameness caused by Escherichia coli. Gross lesions included enlarged and congested spleens and livers, and dilated discolored black or purple duodenal loops. Microscopic lesions included splenic and hepatic congestion. In some birds (freshly killed and fixed immediately), the epithelium at the tips of the duodenal villi was sloughing, but in other birds the villi were intact and normal in appearance. Splenic enlargement, the presence of intranuclear splenic inclusions similar to those found in hemorrhagic enteritis (HE), and the isolation of HE virus from some of the field spleens all indicated that inapparent HE infection often occurs at approximately the same time as this type of colibacillosis. It is therefore believed that HE infection often exacerbates colibacillosis of older poults.     

The effect of infectious bursal disease virus on the immune system of turkeys

In 1979 it was reported that an infectious bursal disease virus (IBDV) isolated from a case of respiratory disease of turkeys differed antigenically from the chicken isolates of this virus. We injected turkey poults with the turkey-originating TY89 and chicken-originating BD/6 isolates of IBDV and studied their effects on antibody production to the virus, serum immunoglobulin G (IgG), antibody response to sheep erythrocytes, in vitro response of peripheral blood lymphocytes to mitogens, and microscopic structure of the bursa of Fabricius. The chicken isolate BD/6 caused a significant decrease in the response to sheep erythrocytes, lower serum IgG, transient decrease in the response of lymphocytes to PHA, and mild microscopic lesions in the bursa of Fabricius. The turkey isolate TY89, however, caused no obvious damage to the immune system of the infected poults. We suggest that a partial and transient functional disorder of the immune system of poults can occur after infection with IBDV originating from chickens, even if the poults exhibited no clinical signs.     

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.