Immune dysfunction following infection with chicken anemia agent and infectious bursal disease virus. II. Alterations of in vitro lymphoproliferation and in vivo immune responses.

To determine the functional impact of alterations in lymphocyte concentrations and ratios following infection with chicken anemia agent (CAA) alone or in combination with infectious bursal disease virus (IBDV) on the immune system of young chickens, in vitro lymphoproliferation assays and in vivo responses to vaccination with several common viral agents were assessed at various time intervals post-inoculation (PI). Concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) stimulation of splenic lymphocytes (SPL) collected from control birds could not be detected until 10-14 days PI. Infection with CAA was characterized by significantly higher PWM stimulation of SPL at 17 days PI and significantly lower PWM stimulation of peripheral blood lymphocytes (PBL) at 14 days PI, compared with uninfected controls. Concanavalin A and PWM stimulation of SPL was significantly increased in birds inoculated with IBDV alone. Lymphocytes harvested from birds inoculated simultaneously with CAA and IBDV had significantly lower responses. Effects on humoral and cell-mediated immunity following CAA and/or IBDV were determined by evaluating vaccination responses to Newcastle disease virus (NDV), fowl pox virus (FPV) and infectious laryngotracheitis virus (ILTV) during the acute phase of CAA infection (2 weeks PI). Vaccination of birds 2 weeks following CAA infection at 1 day of age resulted in decreased protection against NDV (85.7%) and ILTV (7.1%) challenge compared with protection rates in control birds (100% and 53.3% respectively). Infectious bursal disease virus infection was associated with decreased protection against NDV (60%) only. Concomitant infection at 1 day of age resulted in a greater reduction in NDV challenge protection (33.3%), slightly decreased FPV protection (87.5%), increased numbers of persistent FPV vaccination lesions and increased protection against ILTV challenge (71.4%). Vaccination of birds 2 weeks following CAA infection at 2 weeks of age resulted in slightly decreased NDV humoral antibody, development of persistent FPV vaccination lesions (17%) and increased immunity to ILTV challenge compared with control birds (83.3% vs. 66.7%). Chickens inoculated with IBDV alone displayed a more severe depression in NDV antibody titers and only a slight decrease in ILTV protection. Vaccination following concomitant infection at 2 weeks of age resulted in a higher percentage of FPV persistent vaccination lesions (39%) and greatly enhanced immunity to ILTV challenge (100%).     

Advancement in vaccination against Newcastle disease: recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to the limitation in their efficacy, current vaccination strategies against ND need improvements. This study aimed to evaluate a new-generation ND vaccine for its efficacy in providing clinical protection and reducing virus shedding after challenge. Broiler chickens were vaccinated in ovo or subcutaneously at hatch with a turkey herpesvirus-based recombinant vaccine (rHVT) expressing a key protective antigen (F glycoprotein) of Newcastle disease virus (NDV). Groups of birds were challenged at 20, 27, and 40 days of age with a genotype V viscerotropic velogenic NDV strain. Protection was 57% and 81%, 100% and 95%, and 100% and 100% after the subsequent challenges in the in ovo and subcutaneously vaccinated chickens, respectively. Humoral immune response to vaccination could be detected from 3-4 wk of age. Challenge virus shedding was lower and gradually decreased over time in the vaccinated birds compared to the unvaccinated control chickens. In spite of the phylogenetic distance between the NDV F gene inserted into the vector vaccine and the challenge virus (genotype I and V, respectively), the rHVT NDV vaccine provided good clinical protection and significantly reduced challenge virus shedding.     

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.     

The serological responses of chickens to mass vaccination with a live V4 Newcastle disease virus vaccine in the field and in the laboratory 1. Meat chickens

Meat chickens on commercial broiler farms were vaccinated once at 1 to 15 days of age with a live V4 Newcastle disease virus (NDV) vaccine administered by drinking water, aerosol or coarse spray. Hatchmates were housed and similarly vaccinated in laboratory isolation pens. Samples of birds were bled at weekly to fortnightly intervals and the serums tested for haemagglutination inhibiting antibody to NDV. Log2 mean titres of up to 6.26, and assumed protection levels (based on the percentage of birds with log2 titres of 4 or greater) of up to 89%, were obtained in field trials within 4 weeks of vaccination. Differences were observed between the results obtained from parallel field and laboratory trials. The presence of maternal NDV antibody reduced the response to vaccination. The results show that this V4 vaccine can produce an adequate serological response following mass administration to Australian meat chickens housed under commercial conditions.     

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.     

The serological responses of chickens to mass vaccination with a live V4 Newcastle disease virus vaccine in the field and in the laboratory 2. Layer pullets

Layer chickens on a commercial started pullet farm were vaccinated once at 31 to 52 days of age by drinking water or aerosol with live V4 Newcastle disease virus (NDV) vaccine. Flockmates which had been rehoused in laboratory isolation pens shortly beforehand were similarly vaccinated. Samples of birds were bled at intervals and the serums tested for haemagglutination inhibiting antibody to NDV. Log2 mean titres of up to 4.88 and assumed protection levels (based on the percentage of birds with log2 titres of 4 or greater) of up to 81%, were obtained in the field trials within 4 weeks of vaccination. A subsequent laboratory trial further compared the response of different breeds of chicken to different routes of vaccination. Differences were observed between breeds, routes of vaccination, and parallel field and laboratory trials. The results show that this V4 vaccine can produce an adequate serological response following mass vaccination of Australian layer pullets housed under commercial conditions, and that care should be exercised in extrapolating results obtained under laboratory conditions.     

Feasibility of using proteins from Salmonella gallinarum vs. 9R live vaccine for the prevention of fowl typhoid in chickens

Proteins from a field strain of Salmonella gallinarum MSG1 were compared with 9R live vaccine strain for their protection against experimental fowl typhoid in chickens. Proteins from S. gallinarum gave better protection than the 9R live vaccine as measured by clearance of challenge organism from internal organs. Proteins given twice with an adjuvant at 200 micrograms/100 g body weight resulted in 95% protection, compared with 60% protection with 9R given orally. The 9R live vaccine produced more hepatic and splenic lesions and, when administered orally as a single dose, was the least protective (60%). In the group vaccinated subcutaneously with a single dose of 9R without an adjuvant, both the challenge strain and the 9R vaccine strain were isolated from the ovaries of some birds. All chickens vaccinated with 9R strain or with proteins developed antibodies detectable by microagglutination test, and in some vaccinated groups as many as 100% of the birds developed antibody levels detected by seroagglutination.     

A technique for inducing B-cell ablation in chickens by in ovo injection of cyclophosphamide.

The effect of cyclophosphamide (CY) treatment in ovo on avian B and T cells was studied. CY was injected in ovo on the 16th, 17th, and 18th days of incubation. Blood samples were collected periodically from CY-treated and nontreated birds after hatch and were used to measure blood lymphocyte responses to the T-cell and B-cell mitogens, concanavalin A and lipopolysaccharide (LPS), respectively. Additionally, flow cytometric analysis was used to determine the presence of B and T cells in peripheral blood, and birds were vaccinated with Newcastle disease virus (NDV) antigen at 3 wk of age and booster vaccinated at 5 wk of age. CY treatment reduced hatchability by 35%-40%, increased mortality by 3%-5% within the first 2 wk of life, and induced a significant retardation in body weight gains. At 2 wk of age, approximately 50% of CY-treated birds were devoid of B-cell mitogenic responsiveness while demonstrating significant T-cell mitogenic responsiveness. However, B-cell responses were observed at 4 and 6 wk from a small percentage of birds that were originally T-cell responsive and B-cell nonresponsive at 2 wk of age. Flow cytometric analysis of peripheral blood lymphocytes revealed that CY-treated birds had significantly less B cells (or were devoid of B cells) than the corresponding nontreated control birds. However, no significant difference in the T-cell percentage was observed between CY-treated and nontreated birds. CY-treated birds did not produce detectable antibodies specific for NDV during the first and second weeks postvaccination, as demonstrated by hemagglutination inhibition assay. However, antibodies were detected in some CY-treated birds 10 days postbooster. Those antibody-positive birds were found to be the same birds that had subsequently responded to the LPS mitogen on the blastogenesis microassay. This study indicates the importance of monitoring the B- and T-cell responses in CY-treated birds to identify those birds in which B-cell regeneration may have occurred.     

Newcastle disease--seroepidemiologic study of a highly contagious epizootic in poultry and in wild birds in Switzerland

Newcastle disease (ND) is a highly contagious viral disease particularly of domestic poultry. Switzerland is currently declared free from ND. A serosurvey using an ELISA was performed to investigate infections with ND-Virus (NDV) in 260 Swiss laying hen flocks, 169 backyard poultry flocks and 1576 wild birds. For laying hen flocks, a stochastic model was applied to analyse the results from serological testing. Four laying hen flocks were identified as NDV-seropositive, and the true NDV seroprevalence in this population was most likely between 1.3 and 1.5%. NDV antibodies were also detected in five of the 169 backyard poultry-flocks. ND-antibody positive birds were found in 10% of all wild birds examined, with the highest proportions among cormorants, grebes, birds of prey, owls, and swifts. The study indicated that positive flocks must have been in contact with NDV strains causing sub-clinical infection, since no clinical signs had been observed. Moreover, trade of poultry or poultry eggs was considered to be an important factor associated with seropositivity in backyard poultry flocks. Contact to wild birds did not seem to be of major importance.     

Immunosuppression and histopathological changes in the bursa of fabricius associated with infectious bursal disease vaccination in chicken

The effect of the infectious bursal disease (IBD) live virus vaccine on the immune response of chicken was evaluated by the assessment of antibody response following vaccination as well as resistance to challenge with virulent virus. Birds were vaccinated at various ages and later challenged with a heterologous vaccine (NDV) or wild-type IBD virus. The BF was examined for histological changes at regular intervals. Antibody levels to NDV were monitored.

Significantly higher mortality rates were observed in birds vaccinated with IBD vaccine than unvaccinated birds (P < 0.01) following challenge, BF from vaccinated birds showed marked lymphocyte depletion and cellular infiltration with mononuclear cells.

Intraocular NDV (NDV-i/o) vaccine given at day old largely prevented the immunodepressive effect of IBD vaccination on NDV vaccine. Groups that received IBD vaccine on day 14 but no NDV i/o suffered higher mortality (41.2%) and showed lower antibody response than those vaccinated on day 1 (0%) or controls which did not receive IBDV (11.8%).     

Serological and pathological studies of Newcastle disease viruses isolated from caged birds from Southeast Asia

Eleven isolates of Newcastle disease virus (NDV), from caged birds imported from or captured in Southeast Asia in 1979-80, were antigenically divided into five distinct groups. Most of them were distinguishable from more classical NDVs (vaccine B1 strain and Miyadera strain) on the basis of their reactivity to eight monoclonal antibodies against the HN molecule of NDV in hemagglutination-inhibition tests. However, when three representative isolates were evaluated for their biological properties and pathogenicity against 1-day-old chickens, all three were found to be velogenic types that could induce serious symptoms of Newcastle disease and which eventually killed all of the chickens, regardless of the route of infection. There was not any significant correlation between their reactivity patterns with the monoclonal antibodies and their virulence.     

藏药"十三味红花丸"对NDV的抑制试验研究

本试验选用10日龄SPF鸡胚,对藏药“十三味红花丸”进行了人工感染NDV的抑制试验研究,试验结果显示,藏药药液（1g/mL)用2倍、4倍和6倍稀释,对鸡胚感染NDV的保护率分别达100％、90％和80％,比NDV强毒对照组分别提高80、70和60个百分点,NDV强毒接种鸡胚囊液NDV血凝效价明显高于NDV强毒＋藏药药液鸡胚尿囊液NDV血凝效价。试验结果表明,藏药“十三味红花丸”对NDV具有很强的抑制作用。     

Laboratory evaluation of Newcastle disease vaccination programs for broiler chickens

Experiments were conducted to examine the efficacy of various commercial vaccination programs for the prevention of Newcastle disease (ND) in broilers. In all, chicks were from breeders vaccinated against ND via drinking water at 75-day intervals. Vaccination was by company personnel on company premises. In Expt. 1, the initial ND vaccination programs tested were vaccination at 1 day by coarse spray with the Spra-Vac machine or by tracheal instillation with the Beak-o-Vac machine, and vaccination at 7 days via drinking water. In Expts. 2-4, birds initially vaccinated via one of the three previously mentioned methods (Spra-Vac in Expt. 2, Beak-o-Vac in Expt. 3, and drinking water in Expt. 4) were revaccinated against ND by either drinking water or coarse spray with one of two commercial portable machines (ULVA Fan or Spray Master). Serologic and challenge data in Expt. 1 indicated that although broilers vaccinated by any of the three initial routes failed to produce measurable antibody to NDV, all methods resulted in protection against NDV challenge at 35 and 49 days. However, resistance to challenge with virulent ND was greatest in birds initially vaccinated by coarse spray with the Spra-Vac machine. Results in Expts. 2-4 indicated that NDV hemagglutination-inhibition titers were highest and resistance to challenge greatest in birds initially vaccinated at day 1 by coarse spray (Spra-Vac) and then revaccinated at 14 days by coarse spray. There were no differences, however, between the portable coarse spray machines in efficacy in reimmunizing broilers against NDV.     

The role of indigenous wild, semidomestic, and exotic birds in the epizootiology of velogenic viscerotropic Newcastle disease in southern California, 1972-1973.

During an epornitic of velogenic viscerotropic Newcastle disease (VVND) in southern California, free-flying wild birds, captive and free-ranging semidomestic birds, and exotic birds were collected from the quarantine area to determine their role in the epizootiology of the disease. The VVND virus was isolated from 0.04% of 9,446 free-flying wild birds, 0.76% of 4,367 semidomestic birds, and 1.01% of 3,780 exotic birds examined. Three house sparrows and 1 crow directly associated with infected poultry flocks were the only free-flying wild birds from which VVND virus was isolated. Among semidomestic species, ducks, quail, chukars, pheasants, peafowl, pigeons, and doves were found to be infected. Psttacines, pittas, and toucans accounted for 92% of the VVND virus isolations from exotic birds. In addition, domestic Newcastle disease virus (NDV) was isolated from 0.29% of the free-flying wild birds, from 1.65% of the semidomestic birds, and from 0.19% of the exotic birds collected. Hemagglutination-inhibition against domestic NDV was demonstrated in 0.24% of 3,796 wild bird serums, 8.28% of 2,004 semidomestic bird serums, and 3.90% of 231 exotic bird serums tested. Although few free-flying wild birds were infected with VVND virus in this epornitic, the isolation of domestic NDV strains from free-flying wild ducks and mourning doves suggests the potential for transportation of NDV over long distances by migratory birds.     

A survey of Newcastle disease in Swiss laying-hen flocks using serological testing and simulation modelling

Newcastle disease (ND) is a highly contagious viral disease of birds particularly domestic poultry. Switzerland is currently declared free from ND; since vaccination is prohibited, the detection of antibodies against ND virus (NDV) results in the destruction of the respective flock (stamping-out policy). However, in 1995 and 1996, antibody-positive flocks were detected and sporadic ND outbreaks even occurred in Switzerland. Therefore, a serosurvey was done to look for evidence of NDV infections in Swiss laying-hen flocks. The survey was designed to provide 95% confidence of detecting at least one seropositive flock if the flock prevalence were 1%. Thirty blood samples from each of 260 commercial laying-hen flocks were collected during 1996 in a central poultry slaughterhouse. Sera were screened for NDV antibodies with a commercial blocking enzyme-linked immunosorbent assay (ELISA). Samples with a questionable or positive test result were retested with the same ELISA. A stochastic computer model was applied to define a cut-off number of test-positive samples to help to differentiate between true- and false-positive flocks and to estimate the true flock prevalence of infection. Four flocks were identified as NDV-seropositive and the NDV true seroprevalence among commercial laying-hen flocks in Switzerland was most likely between 1.35 and 1.55%. This indicates that Swiss laying-hen and parental flocks with more than 150 animals have been in contact with strains of NDV that cause subclinical infection in chicken, because no clinical symptoms have been observed. In this context, computer simulation was a useful technique to interpret survey results.     

Determination of minimum hemagglutinin units in an inactivated Newcastle disease virus vaccine for clinical protection of chickens from exotic Newcastle disease virus challenge

The potency of inactivated Newcastle disease virus (NDV) vaccines in the United States is currently determined using vaccination and challenge of experimental animals against a velogenic strain of NDV. Because velogenic strains of NDV are now classified as select agents in the United States, all vaccine potency testing must be performed in live animals under biosafety level 3 agriculture conditions. If the minimum amount of inactivated viral antigen required for clinical protection can be determined using other methods, vaccines meeting these criteria might be considered of adequate potency. The linearity of correlation between the hemagglutination (HA) assay measurement and the 50% embryo infectious dose titer ofNDV Hitchner B1 vaccine virus was determined. Correlation between hemagglutinin units (HAU) per vaccine dose, clinical protection, and antibody response was then determined using a vaccinate-and-challenge model similar to Chapter 9 of the U.S. code of federal regulations approved method for vaccine potency testing. The dose providing 50% protection of an in-house water-in-oil emulsion vaccine formulated with inactivated NDV B1 was determined to be between 400 and 600 HAU from two separate trials. A positive correlation (R2 = 0.97) was observed between antibody response and HAU per vaccine dose. Serum antibody responses from vaccinated birds indicate HA inhibition titers >2(5) log2 would provide 100% protection from morbidity and mortality and require a minimum protective dose of 1000 HAU per bird. These are the first studies to examine establishing both a minimum protective HAU content for inactivated ND vaccines and a minimum serologic response necessary to ensure potency.     

A comparison of the onset of protection induced by Newcastle disease virus strain B1 and a fowl poxvirus recombinant Newcastle disease vaccine to a viscerotropic velogenic Newcastle disease virus challenge

Four-week-old specific-pathogen-free white rock chickens were immunized with either a commercial recombinant fowl poxvirus-vectored Newcastle disease vaccine (FPN) expressing the hemagglutinin-neuraminidase and fusion protein genes of Newcastle disease virus (NDV) strain B1 or live NDV B1. Vaccinates and controls were challenged by eyedrop and intranasal (E/I) route with a viscerotropic velogenic NDV at 14 days postvaccination to determine the time of clearance of challenge virus. In a subsequent experiment, chickens were challenged at 3, 6, or 10 days postvaccination to determine the onset of immunity. Chickens that received a recommended field dose (1x) or a 0.01x dose of FP-N subcutaneously (s.c.) and were seropositive by hemagglutination-inhibition test at 14 days postvaccination cleared the challenge virus by 14 days postchallenge. Clinical Newcastle disease and high challenge virus titers in tissues were seen only in seronegative FP-N 0.01x dose vaccinates and controls. In a comparison of vaccination with FP-N (1x, 10(4,9) median tissue culture infective dose) s.c., B1 (10(6) median egg infective dose [EID50]) s.c., or B1 (10(6) EID50) E/I, chickens vaccinated at 6 or 10 days before challenge with all vaccines were protected against clinical disease, but only those vaccinated with B1 E/I 10 days before challenge were protected against infection with the challenge virus. Vaccination at 3 days before challenge with B1 E/I provided early protection, but severe nervous signs developed later and reduced overall protection to 60%, whereas disease in chickens vaccinated with B1 s.c. and FP-N s.c. 3 days before challenge was similar to the challenge controls.     

一株新疆野鸟源新城疫病毒的分离鉴定、F基因克隆及遗传进化分析

为了解新疆野鸟新城疫病毒（Newcastle disease virus,NDV）感染情况,分析其分子特征和基因变异特点,防止新城疫的暴发和蔓延,本试验用9日龄SPF鸡胚进行病毒的分离传代,HA、HI试验和RT-PCR方法对位于"东非-西亚迁徙线"福海县的野鸟进行了NDV检测,分离到1株野鸟源NDV,并命名为NDV/Pintail/CH（XJ）/01/2016。结果表明,该NDV分离株F基因ORF长1 662 bp,编码553个氨基酸,F基因碱性裂解位点序列为¹¹²G-R-Q-G-R-L¹¹⁷,符合弱毒株特征;遗传进化分析显示,NDV/Pintail/CH（XJ）/01/2016与乌克兰鸽源分离株Doneck/3/968、比利时鸭源分离株Simeonovgrad核苷酸同源性均为99.7%,属于NDV ClassⅡ基因Ⅱ型。而上述3株病毒均与疫苗毒La Sota具有较高同源性（99.5%~99.8%）。本研究结果为家禽NDV外流进入自然环境提供了论据。     

Pathogenicity and immunogenicity of different recombinant Newcastle disease virus clone 30 variants after in ovo vaccination

Even though Newcastle disease virus (NDV) live vaccine strains can be applied to 1-day-old chickens, they are pathogenic to chicken embryos when given in ovo 3 days before hatch. Based on the reverse genetics system, we modified recombinant NDV (rNDV) established from lentogenic vaccine strain Clone 30 by introducing specific mutations within the fusion (F) and hemagglutinin-neuraminidase (HN) proteins, which have recently been suggested as being responsible for attenuation of selected vaccine variants (Mast et al. Vaccine 24:1756-1765, 2006) resulting in rNDV49. Another recombinant (rNDVGu) was generated to correct sequence differences between rNDV and vaccine strain NDV Clone 30. Recombinant viruses rNDV, rNDV49, and rNDVGu have reduced virulence compared with NDV Clone 30, represented by lower intracerebral pathogenicity indices and elevated mean death time. After in ovo inoculation, hatchability was comparable for all infected groups. However, only one chicken from the NDV Clone 30 group survived a 21-day observation period; whereas, the survival rate of hatched chicks from groups receiving recombinant NDV was between 40% and 80%, with rNDVGu being the most pathogenic virus. Furthermore, recombinant viruses induced protection against challenge infection with virulent NDV 21 days post hatch. Differences in antibody response of recombinant viruses indicate that immunogenicity is correlated to virulence. In summary, our data show that point mutations can reduce virulence of NDV. However, alteration of specific amino acids in F and HN proteins of rNDV did not lead to further attenuation as indicated by their pathogenicity for chicken after in ovo inoculation.     

Effects of Polyether Ionophores on the Protective Immune Responses of Broiler Chickens against Angara Disease and Newcastle Disease Viruses

Immunization against Angara disease virus (ADV), a serotype 4 avian adenovirus, and Newcastle disease virus (NDV), an avian paramyxovirus serotype 1, is the mainstay of a broiler vaccination programme, while polyether ionophores usually form an essential component of a broiler medication programme in most parts of India and Pakistan. The role of polyether ionophores in the protective immune responses of broiler chickens vaccinated and challenged with ADV and NDV was investigated. A total of 1600 birds were divided into eight groups of 200 birds each. First four groups were vaccinated against NDV and ADV, while the remaining four served as unvaccinated controls. The first 3 groups of birds were administered salinomycin, monensin and cyclophosphamide (CYP), respectively. The last group served as an untreated control. The same treatment schedule was also followed for the next four unvaccinated groups. The post-vaccination and post-challenge serological responses to NDV and ADV, body and lymphoid organ weight gains, post-challenge survival rate and detection of NDV and ADV in the tissues of infected birds were evaluated. Birds administered salinomycin showed a significant stimulation of protective immune responses against both NDV and ADV as compared to the untreated and CYP-treated birds. Monensin also enhanced the protective immune responses against both viruses but the effect was not statistically significant. Thus, it is concluded that monensin and salinomycin augment the anti-NDV and anti-ADV immune responses in broiler chickens, which supports their use in poultry flocks.