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.     

Evidence for bursal involvement in the pathogenesis of hemorrhagic enteritis of turkeys

The pathogenesis of hemorrhagic enteritis in turkey poults infected with hemorrhagic enteritis virus (HEV) at 3 days or at 2 or 5 weeks of age was compared with pathogenesis in poults that had been chemically bursectomized neonatally and exposed to cell-culture-propagated virus at 2 or 5 weeks of age. Conventional poults exposed to HEV at 2 or 5 weeks developed clinical disease, and mortality ranged from 38% to 100%. In addition to the splenic and intestinal lesions usually seen with HEV infection, the pancreas, bursa of Fabricius, and thymus were also affected. In contrast, although they were free from detectable maternal antibody, poults infected with HEV at 3 days of age failed to develop clinical disease or mortality; however, virus was demonstrated by histological and electron microscopic examinations in spleens of these poults. Neonatal chemical bursectomy completely prevented the clinical signs, gross lesions, and mortality induced by HEV in poults at 2 or 5 weeks of age. These findings strongly suggest that an intact bursa is necessary for HEV to induce disease in turkeys.     

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.     

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.     

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.     

Pathogenesis of marble spleen disease in bursectomized and non-bursectomized ring-necked pheasants following oral inoculation with cell-culture-propagated virus.

Seventy-two 13-week-old ring-necked pheasants were inoculated orally with 5.0 x 10(2) tissue-culture infective dose (TCID) of cell-culture-propagated marble spleen disease virus. Inoculated birds exhibited neither mortality nor clinical disease. Gross and histologic lesions were typical of marble spleen disease. The mean splenic weight was significantly (P less than 0.02) higher in inoculated birds than in controls between 6 and 10 days postinoculation (PI). The histologic splenic lesions, which consisted of reticuloendothelial cell hyperplasia, intranuclear inclusions within reticuloendothelial cells, and lymphoid depletion, were most prominent between 6 and 10 days PI. In a second experiment, 1-day-old pheasants were chemically bursectomized by dosing birds with 1.2 mg cyclophosphamide on 3 consecutive days. At 7 weeks of age, 54 bursectomized birds were inoculated orally with 5.0 x 10(2) TCID of marble spleen disease virus. Gross and histologic lesions were detected in one of the inoculated pheasants, but the mean splenic weight was not significantly different from control birds at any time PI. These results are evidence of the role of the bursa of Fabricius in the pathogenesis of marble spleen disease.     

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).     

Effects of photostimulation at 18, 24 and 30 weeks of age on the productivity of female turkeys fed ad libitum or restricted until point of lay.

1. Turkey females were fed ad libitum or restricted from 6 weeks of age to achieve mean body weights of 0.6 of ad libitum fed birds on photostimulation at 18, 24 or 30 weeks of age. Body weight, food intake, egg production and hatchability were recorded to 54 weeks of age. 2. Restricted turkeys were fed ad libitum after first egg or 36 weeks of age. Food intake after 30 weeks of age averaged 170 g/bird/d and was similar for all treatments. At 54 weeks of age, restricted turkeys photostimulated at 24 and 30 weeks were not as heavy as turkeys photostimulated at 18 weeks and ad libitum fed turkeys. 3. A large proportion of restricted turkeys photostimulated at 18 weeks of age did not commence lay until 30 to 40 weeks and a significant number of restricted birds photostimulated at 24 weeks had short laying cycles. Restricted birds photostimulated at 30 weeks came into lay and showed similar persistency of lay to ad libitum fed turkeys. 4. Ad libitum fed turkeys laid 115.0, 122.0 and 101.0 and restricted turkeys 92.4, 99.5 and 103.4 eggs when photostimulated at 18, 24 and 30 weeks, respectively. The number of non-settable eggs was lower in restricted compared with ad libitum fed turkeys and declined with age at photostimulation. 5. Egg size at the end of the experiment was similar for ad libitum fed turkeys and restricted birds photostimulated at 18 weeks but was 2.3 g lower for restricted birds photostimulated at 24 and 30 weeks of age. 6. Hatchability was higher, and the proportion of second quality poults was lower with eggs from restricted turkeys. The mean numbers of poults hatched were 59.9, 75.1 and 66.0 for ad libitum fed and 71.7, 65.7 and 79.4 for restricted turkeys photostimulated at 18, 24 and 30 weeks of age.     

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.     

Effect of field beans and supplemental methionine on growth of turkey poults

Male poults housed at 10 and 15 birds/pen and female poults housed at 15 birds/ pen were fed, from 6 to 10 weeks, on diets containing o, 15, 30 and 30% field beans (Vicia faba L.). The difference between the last two diets was in the level of methio‐nine; one contained 1.6 and the other 1.9%. From 10 to 16 weeks of age birds were fed on diets containing 0 or 20% field beans. At 10 weeks birds given diets with 30% beans were significantly lighter than other poults. At 16 weeks only the poults previously given a diet with 1.9% methionine were significantly lighter than control birds. Males given 0.37 m² of floor space were significantly heavier at 16 weeks than those given 0.25 m².     

Papovavirus induced feather abnormalities and skin lesions in the budgerigar: clinical and pathological findings

Feather abnormalities and skin lesions caused by a papovavirus infection in budgerigars are described. Diseased one to 15 day old birds displayed a lack of nestling down feathers and filoplumes on the head and neck. Survivors older than 15 days exhibited retarded growth and temporary absence of feathers variable from bird to bird. Several birds between 15 and 25 days of age had flight feathers with total absence or marked sparseness of the vanes. After 25 days, feathers began to grow, although the tail and/or some flight feathers of some of the birds remained underdeveloped or absent for several weeks. Several of these affected birds were unable to fly and are called “runners”

Microscopic lesions in the feather follicles of the affected birds less than 15 days of age, were characterized by focal, multifocal or diffuse ballooning degeneration in the lateral and axial plate cells of the barb ridges with the presence of large basophilic or amphophilic intranuclear inclusions in the same cells. Focal areas of ballooning degeneration with intranuclear inclusions were also found in the epidermis. Clinical observations made on these birds are compared with those reported in the literature for French molt.

     

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.     

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.     

Epidemiological and clinical investigations into big liver and spleen disease of broiler breeder hens

SUMMARY The epidemiological and clinical features of big liver and spleen disease (BLS) in flocks on two broiler breeder farms were investigated by serology and gross pathology. The most common necropsy findings on farm 1 were splenomegaly and hepatomegaly, with kidney enlargement in some birds. In one flock (farm 1), a decline in egg production began at 40 weeks of age and lasted for 9 weeks. Seroconversion to BLS antigen was first detected at 45 weeks (3.1% of birds) and increased to 72% at 50 weeks, which coincided with clinical recovery in the flock. Antigen was detected before antibody at 44 weeks and persisted at low incidence (<15%). Farm egg production statistics and serology indicated that the disease affected all flocks on the farm. In three of eight flocks, seroconversion was detected in birds before peak production. The birds in the remaining sheds did not seroconvert or become sick until after peak production. On the second farm, sampling began within a flock already experiencing BLS. Clinical signs and pathology were similar to those seen in flocks on farm 1. However, the lesions that were seen in the pancreas in 15% of birds have not been reported previously. BLS antibody was detected in 78%, and circulating antigen in 14%, of sick birds.     

滴鼻途径感染猪血凝性脑脊髓炎病毒小鼠的免疫动态变化

应用石蜡切片、ELISA等方法,通过对小鼠脾脏病理组织学观察以及脾脏指数、IL-2、IFN-γ和TNF-α等重要细胞因子的检测,研究猪血凝性脑脊髓炎病毒（Hemagglutinatingencephalomyelitisvirus,HEV）感染不同周龄（4、6周龄）BALB／c小鼠后的免疫动态变化特点。结果显示,无论是4周龄还是6周龄小鼠,在被HEV感染后的1～3d,脾小体生发中心增大并在红髓有多量浆细胞出现,第4天脾小体开始缩小,其周边和中央动脉周围有多量淋巴细胞聚集,到第5天脾小体生发中心消失并在其周边和中央动脉周围淋巴细胞增多。另外,脾脏指数和血清中的TNF-α、IFN-γ、IL-4浓度均呈现先升高后降低的趋势,IL-2含量变化不明显,而IL-10含量较少几乎检测不到。结果表明,HEV感染初期,小鼠对侵入的病毒做出一定的免疫应答,但是随着病毒复制,病毒量的增大,小鼠的免疫反应受到抑制。同时不同周龄小鼠的被检指标降低或升高程度及持续时间不同,表明HEV的免疫、发病与小鼠感染年龄之间存在一定相关性。     

Direct lateral transmission of Histomonas meleagridis in turkeys

The lateral transmission of Histomonas meleagridis in turkeys was studied in floor pens without the presence of Heterakis gallinarum. Battery-reared poults (120) were transferred at 2 wk of age to concrete-floored floor pens with fresh pine shavings litter (40/group). One group received no exposure. In other groups, either 10% or 25% of the birds were inoculated per cloaca with cultured H. meleagridis (200,000/bird) and placed in the pens as seeder birds. Inoculated birds died at 10-18 days postinfection (PI) showing typical liver and cecal lesions of histomoniasis. Birds in the high-exposure group died of histomoniasis beginning 16 days PI and continuing to 100% mortality by day 23 PI. Birds in the low-exposure (LE) group died beginning on day 19 PI and continuing through day 31 PI. All but one LE bird alive on day 31 PI had severe liver and cecal lesions of histomoniasis at necropsy. There was no evidence of histomoniasis in unexposed birds. No cecal worms (H. gallinarum) were found at necropsy of dead birds or in unexposed birds at the end of the experiment. Even though H. gallinarum is the only known reservoir for H. meleagridis, these results suggest that lateral transmission of histomoniasis through a flock can occur readily through normal contact between uninfected birds and infected birds and their droppings in the total absence of cecal worms.     

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 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.