The immune response of young turkeys to haemorrhagic enteritis virus infection at different levels and sources of methionine in the diet.

BACKGROUND: Haemorrhagic enteritis (HE) of turkeys was first described in 1937 in the USA, while in Poland it was first diagnosed in 1987. Polish haemorrhagic enteritis virus (HEV) isolates are usually low pathogenic and trigger a subclinical disease. Unfortunately, even the low- pathogenic HEV strains cause severe immunosuppression leading to secondary bacterial infections and huge economic losses. The objective of this study was to evaluate if the influence of Met on HEV infected turkeys immune response can be differentiated by both its level and source. Met is one of the amino acids that not only play a nutritional role but also participate in and regulate key metabolic pathways and immune response. In our study, the birds were assigned to 4 dietary treatments which differed in Met levels (0.55 and 0.78% in weeks 1-4 of age and 0.45 and 0.65% in weeks 5-8 of age, respectively) and sources (DL-methionine (DLM) or DL-methionine hydroxy analogue (MHA)). RESULTS: The HEV added the percentage of CD4+ cells and decreased the percentage of IgM+ cells in the blood, spleen and caecal tonsils (CTs) of turkeys. In addition, it increased the percentage of CD4+CD25+ cells in blood, and interleukin-6 (IL-6) level in plasma. The higher dose of Met led to a significant decrease in the percentages of CD4+, CD8+ and CD4+IL-6+ cell subpopulations in the blood of HEV-infected and uninfected turkeys and to an increase in the percentage of IgM+ B cells in CTs. Turkeys administered feeds with an increased Met content displayed a decrease in plasma IL-6 levels and an increase in plasma IgA levels. CONCLUSIONS: The results of this study indicate that HEV infection impairs the immune function in turkeys. Met content in the feed has a moderate effect on the immune response in HEV-infected turkeys. The source of this amino acid appears not be as important as its dose, because value of the analysed parameters did not differ significantly between turkeys receiving feeds with DLM or MHA. In the uninfected turkeys, the higher by 40% (than recommended by NRC) level of Met in the feeds had a positive effect on humoral immunity parameters.

A DNA prime-protein boost vaccination strategy targeting turkey coronavirus spike protein fragment containing neutralizing epitope against infectious challenge.

The present study was undertaken to determine immune response and protection efficacy of a spike (S) protein fragment containing neutralizing epitopes (4F/4R) of turkey coronavirus (TCoV) by priming with DNA vaccine and boosting with the recombinant protein from the corresponding DNA vaccine gene segment. Turkeys were vaccinated by priming with either one dose (G1-750DP) or two doses (G3-750DDP) of 750µg DNA vaccine expressing 4F/4R S fragment and boosting with one dose of 200µg 4F/4R S fragment. One dose of 100µg DNA vaccine mixed with polyethyleneimine (PEI) and sodium hyaluronate (HA) followed by one dose of 750µg DNA vaccine and one dose of 200µg 4F/4R S fragment were given to the turkeys in group G2-100DPH. After infectious challenge by TCoV, clinical signs and TCoV detected by immunofluorescence antibody (IFA) assay were observed in less number of turkeys in vaccination groups than that in challenge control groups. TCoV viral RNA loads measured by quantitative real-time reverse transcription-PCR were lower in vaccinated turkeys than those in challenge control turkeys. The turkeys in G3-750DDP produced the highest level of TCoV S protein-specific antibody and virus neutralization (VN) titer. Comparing to the turkeys in G1-750DP, significantly less TCoV were detected by IFA in the turkeys in G2-100DPH receiving an extra dose of 100µg DNA mixed with PEI and HA. The results indicated that DNA-prime protein-boost DNA vaccination regimen targeting TCoV S fragment encompassing neutralizing epitopes induced humoral immune response and partially protected turkeys against infectious challenge by TCoV.

Seroprevalence of turkey coronavirus in North American turkeys determined by a newly developed enzyme-linked immunosorbent assay based on recombinant antigen.

Turkey coronavirus (TCoV) causes diarrhea in young turkey poults, but little is known about its prevalence in the field. To address this, the complete nucleocapsid gene was cloned and expressed in Escherichia coli. Expressed nucleocapsid gene produced two distinct proteins (52 and 43 kDa); their specificity was confirmed by Western blotting using two different monoclonal antibodies. Recombinant N protein was purified and used as an antigen to develop an enzyme-linked immunosorbent assay (ELISA) for the serological detection of TCoV that was then validated using experimentally derived turkey serum. The N-based ELISA showed (97%) sensitivity and (93%) specificity for TCoV, which was significantly higher than an infectious bronchitis coronavirus-based commercial test for TCoV. To assess the utility of this recombinant ELISA, 360 serum samples from turkey farms in Ontario, Canada, and 81 serum samples from farms in Arkansas were tested for TCoV-specific antibodies. A high seroprevalence of TCoV was found in turkeys from the Ontario farms with 73.9% of breeders and 60.0% of meat turkeys testing seropositive using the N-based ELISA. Similarly, a high field prevalence was found in the turkeys from Arkansas, for which 64.2% of the serum samples tested seropositive.

Mechanical transmission of turkey coronavirus by domestic houseflies (Musca domestica Linnaeaus).

Domestic houseflies (Musca domestica Linnaeaus) were examined for their ability to harbor and transmit turkey coronavirus (TCV). Laboratory-reared flies were experimentally exposed to TCV by allowing flies to imbibe an inoculum comprised of turkey embryo-propagated virus (NC95 strain). TCV was detected in dissected crops from exposed flies for up to 9 hr postexposure; no virus was detected in crops of sham-exposed flies. TCV was not detected in dissected intestinal tissues collected from exposed or sham-exposed flies at any time postexposure. The potential of the housefly to directly transmit TCV to live turkey poults was examined by placing 7-day-old turkey poults in contact with TCV-exposed houseflies 3 hr after flies consumed TCV inoculum. TCV infection was detected in turkeys placed in contact with TCV-exposed flies at densities as low as one fly/bird (TCV antigens detected at 3 days post fly contact in tissues of 3/12 turkeys); however, increased rates of infection were observed with higher fly densities (TCV antigens detected in 9/12 turkeys after contact with 10 flies/bird). This study demonstrates the potential of the housefly to serve as a mechanical vector of TCV.

Specific mucosal IgA immunity in turkey poults infected with turkey coronavirus.

The objective of this study was to elucidate the kinetics and magnitudes of specific IgA antibody responses in intestines of turkey poults infected with turkey coronavirus (TCV). Turkey poults were orally inoculated with TCV at 10 days of age. Intestinal segment cultures were administered for duodenum, jejunum, and ileum and the IgA antibody responses were analyzed at 1, 2, 3, 4, 6, or 9 weeks post-infection (PI) in two different experiments. The kinetics of virus-specific IgA antibody responses in duodenum, jejunum, and ileum were similar: gradually increased from 1 week PI, reached the peak at 3 or 4 weeks PI, and declined afterward. The virus-specific IgA antibody responses in duodenum, jejunum, and ileum showed negative correlation with duration of TCV antigen in the corresponding locations of intestine with Spearman's correlation coefficient of -0.85 (p=0.034), -0.74 (p=0.096), and -0.75 (p=0.084), respectively. Moreover, the virus-specific IgA antibody responses in serum were positively correlated with that of duodenum (coefficient=0.829, p=0.042), jejunum (coefficient=0.829, p=0.042), and ileum (coefficient=0.771, p=0.072) segment cultures, suggesting that the induction of specific IgA response in serum was predictive of an IgA response in intestine. The results indicate that intestinal mucosal IgA antibodies to TCV are elicited in turkeys following infection with TCV. The local mucosal antibodies may provide protective immunity for infected turkeys to recover from TCV infection.

The effect of immunosuppression on protective immunity of turkey poults against infection with turkey coronavirus.

The objective of the present study was to evaluate the protective effect of humoral and cellular immunities on turkeys infected with turkey coronavirus (TCV). Two trials were conducted with two separate hatches of turkey poults. Turkey's were experimentally immunosuppressed with cyclosporin A (CsA) or cyclophosphamide (CY) and infected with TCV. Prior to infection, treatment with CsA selectively suppressed T cell activity as revealed by 2-3 fold decreased (p < 0.1) lymphocyte proliferation responses to a T cell mitogen, concanavalin A (Con A). Treatment with CY mainly induced B cell deficiency as indicated by significant reductions (p < 0.05) in antibody responses to sheep erythrocytes 7 days after injection. Body weight gain of turkeys treated with CY was significantly lower (p < 0.05) than that of untreated turkeys at 9 days post-infection (PI). Turkeys treated with CY had 1-2 fold higher immunofluorescent antibody assay (IFA) scores for TCV antigens (p < 0.05) in the intestine than untreated turkeys at 9 or 14 days PI. These results suggested that humoral immunity against TCV infection may be important in turkeys.

Humoral and cellular immune responses in turkey poults infected with turkey coronavirus.

The objective of the present study was to elucidate the kinetics of humoral and cellular immune responses of turkey poults infected with turkey coronavirus (TCV). Turkey poults were orally inoculated with TCV at 10 d of age, and the immune responses were analyzed at 1, 3, 7, 14, 21, 28, 42, and 63 d postinfection (PI) in three different experiments. Total Ig to TCV was initially detected at 7 and 14 d PI in Experiments 1 and 3. In addition, Ig gradually increased from 7 to 21 d PI and remained at 80 immunofluroescent antibody assay (IFA) titers or more thereafter. Lymphocyte proliferation responses of spleen cells to concanavalin A were higher in TCV-infected turkeys than in noninfected control turkeys with significant differences (P < 0.05) being noted at 14 and 63 d PI in Experiment 2 and at 3 and 28 d PI in Experiment 3. Strong IFA staining response to TCV antigen was observed in intestines of turkeys at 1, 3, and 7 d PI, and the response declined from 14 to 28 d PI in Experiment 3. In Experiment 3, the IgG isotype antibody response to TCV was markedly increased after 21 d PI and remained high until 63 d PI. The IgM isotype antibody response to TCV was 1.40 and 0.91 at 7 and 14 d PI, respectively. The IgA isotype antibody response to TCV was very low as detected at 7 (0.13), 14 (0.20), and 21 (0.17) d PI. Turkeys infected with TCV had significantly higher (P < 0.05) antibody response to sheep erythrocytes than noninfected controls at 7 d PI. Virus-specific lymphocyte proliferation response of spleen cells was significantly stimulated (P < 0.05) at 63 d PI in Experiment 3. The proportion of the CD4+ subpopulation of T lymphocytes was significantly increased (P < 0.05) at 1, 7, and 21 d PI in Experiment 3. The results indicate that humoral and cellular immunities to TCV are elicited in turkeys following infection with TCV.

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen.

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.

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 naïve 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, naïve 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.

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.

Immunopathogenesis of haemorrhagic enteritis virus (HEV) in turkeys.

Infection of turkeys with the haemorrhagic enteritis virus (HEV), a type II avian adenovirus, results in varying rates of morbidity and mortality. The disease is characterised by splenomegaly, intestinal haemorrhage, sudden death and immunosuppression. The mechanisms of HEV immunopathogenesis and immunosuppression are not fully understood. Recent studies indicate that immune responses play a central role in disease pathogenesis. HEV infects B cells and macrophages and induces necrosis as well as apoptosis in infected and possibly in by-stander cells. The ability of the infected birds to mount an optimum humoral immune response as well as normal macrophage functions such as phagocytosis may be impaired. Elevated numbers of splenic CD4(+) cells during the acute phase of infection may be associated with viral clearance. Types I and II interferons (IFN) and pro-inflammatory cytokines such as interleukin-6 and tumour necrosis-like factors (TNF) are released at the peak of the infection. Cytokines may play a protective as well as a destructive role. While a massive release of proinflammatory cytokines may lead to systemic shock associated with haemorrhagic enteritis and death, release of IFNs may protect turkeys from the disease. Treatment with thalidomide, which is a potent TNF down-regulatory drug, prevented HEV-induced intestinal haemorrhage and treatment with an IFN-inducing chemical prevented HEV-replication and inhibited HEV-induced pathological and histopathological lesions.

Comparative pathogenesis of haemorrhagic enteritis virus (HEV) infection in turkeys and chickens.

The pathogenesis of haemorrhagic enteritis virus (HEV) infection in chickens 3-4 days post-infection was compared with that in turkeys. As expected, infected turkeys showed HEV-specific lesions that included enlargement and mottling of the spleen, as well as haemorrhagic enteritis. In infected chickens, only splenomegaly was observed. The number of HEV-infected cells in the spleen was significantly (P < 0.05) higher in the turkey than in the chicken. In both species, the immunohistochemical labelling of B-cell surface determinants was diminished and the splenic B-cell areas were undetectable after HEV infection. Infection with HEV resulted in an increase in nitric oxide production by macrophages in chickens but not in turkeys.

Alterations in the lymphocytic and mononuclear phagocytic systems of turkey poults associated with exposure to poult enteritis and mortality syndrome.

In vivo and in vitro mononuclear phagocytic system functions, expression of lymphocyte subset cell surface markers in the thymus and bursa of Fabricius, and lymphocyte subset dynamics during the course of poult enteritis and mortality syndrome (PEMS) were examined. PEMS is an acute, transmissible, infectious intestinal disease accompanied by high mortality and morbidity. The etiology of this multifactorial disease remains to be elucidated; however, turkey coronavirus was initially assumed to be one of the primary agents involved. Further investigation demonstrated that turkey coronavirus was not always detectable in poults exhibiting PEMS symptoms, and, thus, PEMS poults began to be identified as positive or negative for turkey coronavirus. In each trial, uninfected hatchmate controls were compared with turkey poults that were contact exposed to PEMS poults at 7 days of age. Following intravenous inoculation, control poults cleared Escherichia coli from their circulation by 60 min, whereas viable E. coli were still present in the circulation of PEMS poults at 60 min postinoculation. Inflammatory response measured by Sephadex-elicited abdominal exudate cell recruitment and the adherence potential of abdominal exudate cells was not significantly different between uninfected and PEMS poults. The percentage of glass-adherent abdominal exudate macrophages was higher in PEMS poults. However, the ability of these macrophages to phagocytize sheep red blood cells and the average number of sheep red blood cells per phagocytic macrophage were both lower compared with uninfected controls. CD4+ expression in thymic tissue of PEMS poults at 9 days postinfection was significantly lower. The CD4+:CD8+ lymphocyte ratio in peripheral blood leukocytes from coronavirus-negative PEMS poults was lower than that from both uninfected and coronavirus-positive PEMS poults at 14 days postinfection. In the spleen, the CD4+:CD8+ lymphocyte ratio was higher in coronavirus-positive PEMS poults as compared with the other treatments. In conclusion, immune system dysfunction in PEMS is associated with impaired mononuclear phagocytic system function and alterations in lymphocyte populations.

Hemorrhagic enteritis virus induced changes in the lymphocyte subpopulations in turkeys and the effect of experimental immunodeficiency on viral pathogenesis.

We examined the changes in the lymphocyte subpopulations in the spleen and peripheral blood of turkeys and the effects of experimental immunodeficiency in the B and T cell compartments on the pathogenesis of hemorrhagic enteritis (HE) in turkeys. Inoculation of turkeys with hemorrhagic enteritis virus (HEV) induced a drop in the relative proportions of IgM bearing cells on Day 2, 3, and 9 post-infection and an elevation in the relative proportions of CD4+ cells on Day 4 and 6 post-infection. Elevated levels of CD8+ cells were observed in the infected turkeys only on Day 16 after infection. Marked depletion of IgM+ cells may play a role in immunodepression caused by HEV. Cyclophosphamide (CY) treatment induced B cell deficiency in turkeys severely impaired HEV replication in the spleen suggesting that B lymphocytes are important for viral replication. Cyclosporin A (CsA) selectively impaired T cell mitogenesis and protected the turkeys against HEV-induced intestinal hemorrhages. CsA did not prevent viral replication in the spleen or the associated splenomegaly. This result suggested that T cell immunity may be important for intestinal hemorrhaging induced by HEV.

Response of specific-pathogen-free turkeys to vaccines derived from marble spleen disease virus and hemorrhagic enteritis virus.

Tissue-culture-propagated marble spleen disease virus (MSDV-TC) and two preparations of spleen homogenate (MSDV-SH and MSDV-SH-TC) were compared as anti-hemorrhagic enteritis virus (HEV) vaccines in specific-pathogen-free turkeys. Both types of vaccines spread horizontally among turkeys, induced anti-HEV antibodies, and protected turkeys against challenge with virulent HEV. Antibody development and horizontal spread of virus occurred earlier in turkeys given MSDV-SH or MSDV-SH-TC than in those given MSDV-TC. Virulent HEV was serially passed in MDTC-RP19 cells. The 30th passage virus (HEV-P30) was nonpathogenic for turkeys but was immunogenic. Turkeys exposed to HEV-P30 had viral antigen in the spleen, developed neutralizing antibodies, and resisted virulent HEV. The principal difference between MSDV-TC and HEV-P30 vaccines was that MSDV-TC caused well-defined splenomegaly in turkeys, whereas HEV-P30 protected turkeys without causing spleen enlargement.

Evaluation of cell culture propagated and in vivo propagated hemorrhagic enteritis vaccines in turkeys.

The immune response of turkeys to a liquid, was compared with a previously frozen, cell culture propagated hemorrhagic enteritis (HE) vaccine. The liquid cell culture propagated HE vaccine was able to induce 100% seroconversion in turkeys 4 weeks after being vaccinated at 3.5 weeks of age; however, the previously frozen cell culture propagated HE vaccine induced 80% seroconversion 4 weeks post vaccination (P < 0.05). The average seroconversion in turkey flocks administered the liquid cell culture propagated HE was 97% in comparison with 98.5% in flocks given the splenic vaccine (P > 0.05). The complete absence of HE antigens in spleens of birds 5 days after being challenged with the virulent HE virus (40,000 TCID50 per bird) at an age of 9.5 weeks, was used as a model for successful protection against HE disease. The HE antigens were absent from spleens of all challenged birds that were previously vaccinated by the liquid cell culture propagated HE vaccine or splenic vaccine.

Hemorrhagic enteritis of turkeys.

Hemorrhagic enteritis (HE), an economically important disease of turkeys is caused by a type II adenovirus. The virus is ubiquitous and is liable to infect most field turkeys. In unprotected turkey flocks, infection with virulent hemorrhagic enteritis virus (HEV) may result in variable mortality and immunodepression. Turkeys younger than 2-4 weeks of age are resistant to clinical HE. This age-related resistance is expressed in the presence or absence of maternal antibodies against HEV. Clinical disease is characterized by HE and splenomegaly. The virus causes intranuclear inclusions in the reticuloendothelial cells. Bursectomy or splenectomy abrogate clinical HE. Field data and laboratory studies indicate that HEV causes immunodepression in the humoral as well as the cellular immune functions of turkeys. The mechanism of immunodepression is not known.

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.

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).

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.