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.     

Immunization of turkeys with a DNA vaccine expressing either the F or N gene of avian metapneumovirus

In this study we compared protection by DNA vaccination with the F (pCMV-F) or N (pCMV-N) gene from avian metapneumovirus (aMPV) in turkeys. One-week-old turkey poults received two intramuscular injections 2 wk apart. Birds were challenged with a turkey-embryo-adapted aMPV at 5 wk of age. Birds vaccinated with pCMV-F had decreased clinical signs of disease as well as significantly reduced virus load in tracheal swabs compared with birds vaccinated with pCMV-N or unvaccinated control birds. Serum neutralizing antibodies were significantly higher in birds receiving pCMV-F compared with all other groups. These results indicate that DNA vaccination with the F, but not N, gene of aMPV can induce significant protection against aMPV infection.     

The fluorescent antibody and indirect fluorescent antibody assays for diagnosing stunting syndrome of turkeys

The fluorescent antibody (FA) assay was developed for detecting the stunting syndrome agent (SSA) from intestinal tissue. Similarly, the indirect fluorescent antibody (IFA) assay was developed for detecting serum antibodies to SSA. Convalescent antiserum from turkeys orally immunized with SSA was found to be the primary antibody of choice for the FA assay. Intestinal jejunal samples from poults inoculated 3-4 days postinoculation (DPI) was found to be the best antigen source for the IFA assay. SSA was detected from the intestinal tracts of experimentally inoculated birds at 2 DPI with highest level of reactivity at 3 DPI by the FA assay. After 4 DPI the level of SSA infectivity of the intestines waned to low levels. Serum antibody was detected from experimentally inoculated birds as early as 7 DPI with all birds tested seroconverting by 12 DPI. These assays should prove useful for future studies concerning stunting syndrome.     

Mucosal vaccination with formalin-inactivated avian metapneumovirus subtype C does not protect turkeys following intranasal challenge

Studies were performed to determine if mucosal vaccination with inactivated avian metapneumovirus (aMPV) subtype C protected turkey poults from clinical disease and virus replication following mucosal challenge. Decreases in clinical disease were not observed in vaccinated groups, and the vaccine failed to inhibit virus replication in the tracheas of 96% of vaccinated birds. Histopathologically, enhancement of pulmonary lesions following virus challenge was associated with birds receiving the inactivated aMPV vaccine compared to unvaccinated birds. As determined by an enzyme-linked immunosorbent assay (ELISA), all virus-challenged groups increased serum immunoglobulin (Ig) G and IgA antibody production against the virus following challenge; however, the unvaccinated aMPV-challenged group displayed the highest increases in virus-neutralizing antibody. On the basis of these results it is concluded that intranasal vaccination with inactivated aMPV does not induce protective immunity, reduce virus shedding, or result in decreased histopathologic lesions.     

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.     

Effect of cyclophosphamide immunosuppression on the immunity of turkeys to viral rhinotracheitis.

Turkey poults, free of antibodies to turkey rhinotracheitis (TRT) virus were treated with cyclophosphamide on days 1, 2 and 3 after hatching and vaccinated by eyedrop when 10 days old with a Vero cell-attenuated preparation of TRT virus. No ELISA antibodies to TRT virus developed in the sera of these poults but they were as resistant to virulent virus challenge 21 days later as vaccinated groups which were not cyclophosphamide-treated but produced humoral antibodies. Following challenge with virulent virus at 31 days old cyclophosphamide-treated unvaccinated poults developed a more severe clinical response than untreated birds and had higher virus titres in tracheal swabs. The findings show that the respiratory tract of turkeys may be resistant to TRT despite the absence of ELISA antibodies in the serum.     

Protective immunity against Newcastle disease: the role of cell-mediated immunity

The role of cell-mediated immunity (CMI) in protection of birds from Newcastle disease was investigated by two different strategies in which only Newcastle disease virus (NDV)-specific CMI was conveyed without neutralizing antibodies. In the first strategy, selected 3-wk-old specific-pathogen-free (SPF) birds were vaccinated with either live NDV (LNDV), ultraviolet-inactivated NDV (UVNDV), sodium dodecyl sulfate-treated NDV (SDSNDV), or phosphate-buffered saline (PBS) (negative control) by the subcutaneous route. Birds were booster vaccinated 2 wk later and challenged with the velogenic Texas GB strain of NDV 1 wk after booster. All vaccinated birds had specific CMI responses to NDV as measured by a blastogenesis microassay. NDV neutralizing (VN) and hemagglutination inhibition (HI) antibody responses were detected in birds vaccinated with LNDV and UVNDV. However, birds vaccinated with SDSNDV developed antibodies that were detected by western blot analysis but not by the VN or HI test. Protection from challenge was observed only in those birds that had VN or HI antibody response. That is, birds with demonstrable CMI and VN or HI antibody response were protected, whereas birds with demonstrable CMI but no VN or HI antibody response were not protected. In the second strategy, birds from SPF embryos were treated in ovo with cyclophosphamide (CY) to deplete immune cells. The birds were monitored and, at 2 wk of age, were selected for the presence of T-cell activity and the absence of B-cell activity. Birds that had a significant T-cell response, but not a B-cell response, were vaccinated with either LNDV, UVNDV, or PBS at 3 wk of age along with the corresponding CY-untreated control birds. The birds were booster vaccinated at 5 wk of age and were challenged with Texas GB strain of NDV at 6 wk of age. All birds vaccinated with LNDV or UVNDV had a specific CMI response to NDV, VN or HI NDV antibodies were detected in all CY-nontreated vaccinated birds and some of the CY-treated vaccinated birds that were found to have regenerated their B-cell function at 1 wk postbooster. The challenge results clearly revealed that CY-treated birds that had NDV-specific CMI and VN or HI antibody responses to LNDV or UVNDV were protected, as were the CY-nontreated vaccinated birds. However, birds that had NDV-specific CMI response but did not have VN or HI antibodies were not protected from challenge. The results from both strategies indicate that specific CMI to NDV by itself is not protective against virulent NDV challenge. The presence of VN or HI antibodies is necessary in providing protection from Newcastle disease.     

Protective immunity against Newcastle disease: the role of antibodies specific to Newcastle disease virus polypeptides

Studies were performed to determine if passive immunization with hyperimmune sera generated to specific Newcastle disease virus (NDV) proteins conferred protection against virus challenge. Six groups of 3-wk-old chickens were passively immunized with antiserum against either hemagglutinin-neuraminidase/fusion, (HN/F) protein, nucleoprotein/phosphoprotein (NP/P), Matrix (M) protein, a mixture of all NDV proteins (ALL), intact ultraviolet-inactivated NDV (UVNDV), or negative sera. Blood samples were collected 2 days postimmunization, and the birds were challenged with Texas GB strain of NDV. Antibody titers were detected from those recipient birds that had received the antisera against the HN/F, ALL, or UVNDV by a hemagglutination inhibition test, an enzyme-linked immunosorbent assay (ELISA), and a virus neutralization test. Antibodies were detected only by the ELISA from the birds that had received antisera against NP/P and M protein. Antibody titers in the recipient birds dropped by two dilutions (log2) after 2 days postinjection. Birds passively immunized with antisera against HN/F, ALL, and UVNDV were protected from challenge, whereas chickens passively immunized with antisera against NP/P and M protein and specific-pathogen-free sera developed clinical signs of Newcastle disease. The challenge virus was recovered from the tracheas of all passively immunized groups. The presence of neutralizing antibodies to NDV provided protection from clinical disease but was unable to prevent virus shedding from the trachea.     

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.     

Infectious bursal disease virus and Alcaligenes faecalis infections in turkeys

To determine if infectious bursal disease virus (IBDV) augments alcaligenes rhinotracheitis (ART), turkey poults were exposed to IBDV, Alcaligenes faecalis, or both IBDV and A. faecalis. In five experiments, poults exposed to IBDV alone exhibited neither signs of disease nor histopathologic lesions. Serum antibodies to IBDV were detected in poults exposed to this virus by inoculation and by direct contact with inoculated birds. Signs of ART were observed 4 to 6 days following exposure to A. faecalis. Clinical signs of ART and histopathologic lesions in the upper respiratory tract of poults exposed to both IBDV and A. faecalis were similar to those observed in poults exposed to A. faecalis alone.     

Pathogenicity of embryo-adapted serotype 2 OH strain of infectious bursal disease virus in chickens and turkeys

The pathogenicity of serotype 2 OH strain of infectious bursal disease virus (IBDV) to specific-pathogen-free (SPF) chicken embryos and 2-wk-old SPF chickens and turkey poults was investigated. The virus was pathogenic for chicken embryos after five passages as evidenced by pathologic changes in inoculated embryos. The embryo-adapted virus was not pathogenic for 2-wk-old SPF chickens and turkey poults as indicated by lack of clinical signs, gross or microscopic lesions in the bursa of Fabricius of inoculated birds. Bursa-to-body-weight ratios of the inoculated chickens and turkey poults were not significantly different from those of uninoculated controls. Virus-neutralizing antibodies to serotype 2 IBDV were detected in inoculated chickens and turkeys. Results of this study indicated that the embryo-adapted serotype 2 OH IBDV isolate that is pathogenic for chicken embryos is infectious but not pathogenic in chickens and turkeys.     

The resistance of meat chickens vaccinated by aerosol with a live V4 Newcastle disease virus vaccine in the field to challenge with a velogenic Newcastle disease virus

Meat chickens housed on a commercial broiler farm in Australia were vaccinated once at 10 to 11 days-of-age by aerosol with live V4 Newcastle disease virus (NDV) vaccine. Groups of vaccinated and unvaccinated birds were flown to Malaysia, where they were challenged with a virulent strain of NDV. Survival rates in vaccinated chickens challenged 7, 14, 21 or 31 d after vaccination were 0.47, 0.77, 0.97 and 0.92, respectively. All unvaccinated chickens died due to Newcastle disease (ND) following challenge. Chickens in Australia and Malaysia were bled and the serums tested for haemagglutination-inhibiting (HI) antibody to NDV. Many vaccinated birds with no detectable antibody, and all birds with a log2 titre of 2 or greater, survived challenge. The results showed that this V4 vaccine induced protective immunity in a significant proportion of chickens within 7 d of mass aerosol vaccination. This early immunity occurred in the absence of detectable circulating HI antibody. Non-HI antibody mediated immunity continued to provide protection up to 31 d after vaccination. Almost all vaccinated birds were protected within 3 w of vaccination. It is concluded that the V4 vaccine is efficacious and could be useful during an outbreak of virulent ND in Australia.     

Use of an enzyme-linked immunosorbent assay to measure antibody levels in turkey breeder hens, eggs, and progeny following natural infection or immunization with a commercial Bordetella avium bacterin

Twelve large white turkey hens were immunized with a commercially available Bordetella avium bacterin. Hens and eggs were tested using an enzyme-linked immunosorbent assay (ELISA) to determine the response to the bacterin. Three hundred poults were then obtained from two commercial flocks, the hens of one flock having been immunized with the same bacterin used on the group of 12 turkeys. Titers of the poults were monitored for 7 weeks, and poults were challenged by exposure to infected poults at 1, 7, 14, and 21 days post-hatch. Hens produced an antibody response following immunization, with a parallel antibody response being detected in eggs. Maternal antibodies were present in poults from immunized hens. Poult titers declined to near the level of poults from unimmunized hens by 14 days of age. Poults from immunized hens challenged at 1 and 7 days were resistant to development of clinical disease and gross lesions, whereas all poults from unimmunized hens exhibited clinical signs and gross lesions. After 14 days, the resistance of both groups to development of clinical disease, became near equal, neither group being affected as severely as the unimmunized hens challenged at days 1 and 7. Six commercial turkey breeding flocks and their progeny that had not been vaccinated for B. avium and had no history of B. avium infection were evaluated with the B. avium ELISA. There were variations between the flocks, with poult titers reflecting those found in the hens.     

Protection of turkey poults from Bordetella avium infection and disease by pili and bacterins

The role of pili in protection against Bordetella avium infection in turkey poults was studied. An isolate that produced the largest number of pili under growth conditions developed in our laboratory was used for preparation of pili and bacterin and for challenge. The pili were isolated, purified, examined by electron microscopy, and tested for purity by gel electrophoresis. Poults were vaccinated with oil-adjuvant pili, formaldehyde- or merthiolate-inactivated bacterins, or a commercial bacterin. Poults were vaccinated once or twice subcutaneously at different ages and challenged intranasally with a pathogenic B. avium isolate 5 days following the last vaccination. A few vaccinated birds had very mild clinical signs. B. avium was isolated from the sinuses of a few vaccinated birds, and growth was scanty. The mean colony counts from tracheal sections was significantly higher (P less than 0.1) in unvaccinated challenged poults than in vaccinated challenged poults. It is postulated that B. avium pili are important immunogens in turkey poults.     

Pathogenicity of Listeria monocytogenes Scott A after oral and oculonasal challenges of day-old turkey poults

Listeria monocytogenes is a ubiquitous, environmental pathogen that has contaminated poultry ready-to-eat products resulting in large-scale recalls. Research is needed to determine the source of product and processing plant contamination with L. monocytogenes. The purpose of this study was to compare the oral and oculonasal routes of infection on the pathogenicity of L. monocytogenes in turkey poults under different housing conditions. One-day-old turkey poults were challenged by either route with the Scott A strain of L. monocytogenes and placed either in paper-lined battery-brooder cages for 1 wk or in floor pens on fresh pine-shaving litter. On day 7, birds challenged in battery cages were transferred to floor pens. Challenge by the oculonasal route resulted in higher mortality (P = 0.05) and lower body weights (P < 0.0001) compared with both nonchallenged controls and those challenged by the oral route. Birds contained in battery cages for 1 wk had higher mortality (P = 0.002) and higher body weights (P < 0.0001) compared with floor-pen-reared birds. Using direct plating, the challenge strain was isolated from the gall bladder, brain, and knee joint of only one dead poult challenged by the oculonasal route. These results suggest that day-old turkey poults may be more susceptible to an oculonasal challenge with L. monocytogenes than to an oral challenge and that containment in battery cages for the first week increased contact exposure to the challenge.     

Effect of passive immunity on the development of a protective immune response against bovine viral diarrhea virus in calves

OBJECTIVE: To determine whether passively acquired antibodies prevent development of a protective immune response to live virus in calves. ANIMALS: 18 calves. PROCEDURES: Calves were caught immediately after birth and tested free of bovine viral diarrhea virus (BVDV) and serum antibodies against BVDV. Within 48 hours, 12 calves were fed colostrum that contained antibodies against BVDV and 6 calves received BVDV antibody free milk replacer. Three milk replacer fed and 6 colostrum fed calves were exposed to virulent BVDV2-1373 at 2 to 5 weeks of life when passively acquired serum antibody titers were high. After serum antibody titers against BVDV had decayed to undetectable concentrations (at 7 to 9 months of age), the 3 remaining milk replacer fed calves, 6 colostrum fed calves previously exposed to BVDV2-1373, and 6 colostrum fed calves that had not been exposed to the virus were inoculated with BVDV2-1373. RESULTS: Passively acquired antibodies prevented clinical disease in inoculated colostrum fed calves at 2 to 5 weeks of life. Serum antibody titers did not increase in these calves following virus inoculation, and serum antibody titers decayed at the same rate as in noninoculated colostrum fed calves. Inoculated colostrum fed calves were still protected from clinical disease after serum antibody titers had decayed to nondetectable concentrations. Same age colostrum fed calves that had not been previously exposed to the virus were not protected. CONCLUSIONS AND CLINICAL RELEVANCE: A protective immune response was mounted in calves with passive immunity, but was not reflected by serum antibodies titers. This finding has implications for evaluating vaccine efficacy and immune status.     

Lack of protection against Bordetella avium in turkey poults exposed to B. avium-like bacteria

Six laboratory experiments were designed to determine whether poults infected with the nonpathogenic Bordetella avium-like (BAL) bacteria would develop immunity to B. avium (BA), the causative agent of turkey coryza. The BAL bacteria were isolated from poults given that organism, but few colonies were observed by 3 weeks postexposure. No serum-agglutinating antibody to the BAL bacterium was detected in poults exposed to that organism. Poults exposed to BAL bacteria either once or twice at different ages were not protected from infection or disease following experimental challenge between 1 and 7 weeks of age with pathogenic BA.