LNA probe-based real-time RT-PCR for the detection of infectious bronchitis virus from the oviduct of unvaccinated and vaccinated laying hens

In the present study, LNA-probe based real-time PCR was designed for the detection and absolute quantification of infectious bronchitis virus (IBV) from the oviduct of unvaccinated and vaccinated hens after IBV challenge. Using a recombinant plasmid standard, the detection limit of the reaction was found to be 10 copies and independent assay runs showed reproducible Ct values. Amongst the unvaccinated hens, the virus could be detected between 6 and 20 days post-infection (p.i.), with a peak of viral load between 10 and 14 days p.i. The virus was also detectable in the oviduct of vaccinated, challenged hens although the viral load was much lower compared to the viral load in the oviduct of unvaccinated, challenged hens. This indicates that rearing phase vaccination can offer significant protection of the fully functional oviduct against a pathogenic strain of IBV. The present test will be useful for the rapid identification of IBV directly from clinical samples. Most vaccination trials investigating the efficacy of vaccines for layer and breeder hens have been conducted based on the respiratory tract response. Evaluation of viral load from the oviduct of vaccinated and unvaccinated hens is an efficient method for assessing oviduct protection in commercial laying hens.     

Vaccination against histomonosis prevents a drop in egg production in layers following challenge

The effect of attenuated Histomonas meleagridis on pullets was investigated and the protection of vaccinated adult laying hens against a severe challenge was studied in the same experimental setting. Four groups of 25 pullets were set up at 18 weeks of life and birds in two groups were vaccinated with in vitro-attenuated H. meleagridis. Chickens in two groups (vaccinated and non-vaccinated) were challenged 5 weeks later with virulent histomonads, while the remaining groups were retained until termination of the study 11 weeks post vaccination. Vaccination of pullets did not have any impact on their subsequent performance. Egg production of non-vaccinated but challenged birds dropped significantly (P≤0.05) between 2 and 4 weeks post challenge (p.c.) to 58.7%, compared with 90% in control chickens. At 4 weeks p.c., the drop in egg production in vaccinated and challenged birds was significantly lower (P=0.02) than in non-protected layers. Pathological changes were found only in challenged birds 2 and 6 weeks p.c. Several non-vaccinated birds showed severe lesions in the caeca with sporadic involvement of the liver and atrophy of the reproductive tract. Vaccination prior to challenge reduced the incidence of pathological findings. For the first time, vaccination of pullets with in vitro-attenuated histomonads could be shown to be an effective and safe prophylactic tool to prevent a severe drop in egg production of commercial layers following experimental infection.     

Studies with a bivalent infectious bronchitis killed virus vaccine

Haemagglutination inhibition and virus neutralisation antibody responses of chickens given different vaccination programmes were compared. This was followed by a further experiment in which variously vaccinated laying hens were challenged at 30 weeks of age with two strains of infectious bronchitis virus of the "variant" Dutch D207 serotype. Chickens were given primary vaccinations to different strains of infectious bronchitis live virus during rearing and then injected at 16 weeks of age with inactivated oil adjuvanted virus vaccines prepared from either M41, GV101 or both viruses combined (bivalent vaccine). Antibody titres to M41 infectious bronchitis virus were high, and to D207 serotype low. in birds given Mass type vaccines only. In birds given an initial 'priming' with Mass type live vaccine and then 'boosted' with bivalent killed vaccine, high haemagglutination inhibition and virus neutralisation antibody levels against both the M41 and D207 serotypes of infectious bronchitis virus were stimulated. In another experiment, the ability of laying hens vaccinated according to this programme, to withstand challenge with two strains of virulent infectious bronchitis virus of the D207 serotype, was tested. Protection of egg production in vaccinated hens was found to be good and in all groups correlated with the individual hen haemagglutination inhibition titre at the time of challenge. The significance of these results with regard to the use of killed virus vaccines in laying hens and to the necessity to develop live virus vaccines from 'variant' strains of infectious bronchitis virus is discussed.     

Vaccination of chickens decreased Newcastle disease virus contamination in eggs

Newcastle disease is an important health issue of poultry causing major economic losses and inhibits trade worldwide. Vaccination is used as a control measure, but it is unknown whether vaccination will prevent virus contamination of eggs. In this study, hens were sham-vaccinated or received one or two doses of inactivated LaSota vaccine, followed three weeks later by virulent Newcastle disease virus (NDV) challenge. Eggs were collected daily and shell, albumen and yolk were subjected to virus isolation, as were oral and cloacal swabs at 2 and 4 days post-challenge (dpc). A second experiment evaluated the distribution of the virus in the reproductive tract of non-vaccinates. All vaccinated chickens survived challenge, and the levels of virus shed from cloacal swabs were decreased significantly when compared to shams. In non-vaccinated hens, virus was detected in the ovary and all segments of the oviduct. Yolk, albumen and eggshell surface from eggs laid at day 4 and 5 post-infection by sham-vaccinated hens were positive for NDV, but eggs from LaSota vaccinated hens lacked virus in internal egg components (i.e. yolk and albumen) and had reduction in the number of positive eggshell surfaces. These results indicate virulent NDV can replicate in the reproductive tract of hens and contaminate internal components of eggs and eggshell surface, but vaccination was able to prevent internal egg contamination, reducing eggshell surface contamination, and reducing shedding from digestive and respiratory tracts in virulent NDV challenged hens.     

Oral vaccination of 1-day-old turkeys with in vitro attenuated Histomonas meleagridis protects against histomonosis and has no negative effect on performance

One-day-old turkey poults were vaccinated against histomonosis (syn. histomoniasis) via the oral route by application of in vitro attenuated Histomonas meleagridis. Subsequently, two different groups composed of 14 birds each were challenged cloacally with highly virulent histomonads after 2 or 4 weeks. Two additional groups of non-vaccinated birds were infected with the challenge inoculum at the same time points. In addition, a group of 19 birds, of which 14 were vaccinated but not challenged, were kept for clinical and serological examinations. Non-vaccinated and non-challenged birds (n=10) represented the negative control group. All non-vaccinated but infected birds and 10 out of 14 vaccinated turkeys challenged 2 weeks post vaccination (w.p.v.) contracted severe histomonosis. Turkeys challenged 4 w.p.v. and all remaining birds used in this experiment did not show any pathognomonic clinical signs. In addition, no adverse effect regarding the weight gain could be observed in birds that were vaccinated but not challenged. The excretion of attenuated and virulent live histomonads was observed very infrequently by re-isolation, but transmission to in-contact birds was very efficient. Presence of antibodies was first noticed 3 w.p.v. and antibody levels remained above the cut-off value until termination of the experiment at 16 w.p.v. The present experiment demonstrates for the first time the potential efficacy of in vitro attenuated histomonads used as an orally applied vaccine to 1-day-old turkeys for protection against fatal histomonosis without affecting performance.     

Avian pneumovirus infection of laying hens: Experimental studies

Administration of a virulent strain of avian pneumovirus (APV) to specific pathogen free laying hens by the oculonasal route failed to induce a drop in egg production or any adverse effects on eggshell quality. However, intravenous (i.v.) inoculation of the same strain caused a substantial drop in egg production and a high incidence of soft and thin-shelled eggs. Some respiratory signs were also observed and the hens appeared sick, with diarrhoea being observed in approximately one-half of the hens between 4 and 11 days post-inoculation (p.i.). APV antigen was detected in the oviduct epithelium up to 9 days p.i. This challenge model was then used to investigate the efficacy of live attenuated turkey rhinotracheitis (TRT) vaccine administered alone at 1 day old, or an inactivated TRT vaccine (at 16 weeks), or a combined programme using both vaccines, in protecting against this challenge. Neither the live nor the inactivated vaccine alone protected against clinical signs (respiratory infection or diarrhoea). However, the inactivated, but not the live, vaccine did protect against the effect of the i.v. challenge on laying performance. In contrast, the combined vaccination programme protected completely against both clinical signs and poor egg-laying performance. This protection lasted until at least 60 weeks of age. On the basis of the results with this experimental model, it is concluded that the use of live priming followed by administration of inactivated TRT vaccine is necessary to provide complete protection of laying chickens against APV challenge.     

Efficacy and safety of an attenuated live QX-like infectious bronchitis virus strain as a vaccine for chickens

The attenuation of infectious bronchitis (IB) QX-like virus strain L1148 is described. The virus was passaged multiple times in embryonated specific pathogen free (SPF) chicken eggs, and at different passage levels samples were tested for safety for the respiratory tract and kidneys in 1-day-old SPF chickens. There was a clear decrease in pathogenicity for the respiratory tract and kidneys when the virus had undergone a large number of passages. Passage level 80 was investigated for safety for the reproductive tract in 1-day-old and 7-day-old SPF chickens. In 1-day-old chickens, 12.5% of the vaccinated birds had macroscopic lesions. No lesions were observed if the chickens had been vaccinated at 7 days of age. Passage level 80 was investigated for its ability to spread from vaccinated to non-vaccinated chickens and for dissemination in the body. The virus was able to spread from vaccinated chickens to groups of non-vaccinated chickens, and in the vaccinated birds the virus was found frequently in oro-pharyngeal and cloacal swabs. A fragment of the hypervariable region of the S1 protein of passage level 80 was sequenced and revealed nucleotide changes resulting in two amino acid substitutions. Passage level 80 was given additional passages to levels 82 and 85. Both passage levels were tested for efficacy in SPF chickens and passage level 85 was tested for efficacy in commercial chickens with maternally derived antibodies (MDA) against a challenge with QX-like strain IB D388. In both SPF chickens and chickens with MDA, the vaccines based on strain IB L1148 were efficacious against challenge.     

Infectious bronchitis in laying hens: The relationship between haemagglutination inhibition antibody levels and resistance to experimental challenge

In three separate and unrelated experiments, in which vaccinated hens were challenged with virulent infectious bronchitis virus, the ability of individual hens to maintain egg production was related to their serum haemagglutination inhibition antibody titre at the time of challenge. It was found that, regardless of the vaccination programme used, the ability of laying hens to withstand infectious bronchitis virus challenge, as measured by the effect upon their egg production, is directly related to individual antibody titre at the time of challenge. In all three experiments, birds with antibody titres of >/=8 Iog2 (n (=) 82) did not show a significant reduction in egg production after challenge while those with titres within the range 5-7 log(2) inclusive (n = 126), over a period of 3 or 4 weeks after challenge, showed a significant reduction in their rate of egg lay, viz: 0.38, 0.33 and 0.47 eggs per hen per week, respectively and those with titres <4 log(2) (n = 101) showed, over the same time period, a reduction of 1.0, 0.45 and 1.16 eggs per hen per week, respectively. The ability of different vaccination programmes to stimulate uniformly high antibody responses to infectious bronchitis virus, and hence good overall protection of egg production was compared. It is concluded that the programme of choice is first to vaccinate the birds with a highly attenuated strain of live infectious bronchitis vaccine during rearing (H120), followed by the injection of a potent killed oil emulsion adjuvant vaccine at point-of-lay. The ability of the less attenuated H52 strain of live infectious bronchitis vaccine to interfere with response to killed vaccine was demonstrated in two of the three experiments. In both cases this interference was accompanied by an increased susceptibility of the hens to the effect of infectious bronchitis virus challenge on egg production.     

The efficacy of Mycoplasma gallisepticum K-strain live vaccine in broiler and layer chickens

The efficacy of a live Mycoplasma gallisepticum (MG) vaccine candidate (K-strain) was compared to commercially available vaccines in broiler-type chickens (Trial 1) and layer-type chickens (Trial 2). In Trial 1, three-week-old broiler-type chickens were vaccinated via aerosol with K-strain or an F-strain vaccine. The vaccinated chickens and 10 non-vaccinated controls were subsequently challenged with virulent R-strain via aerosol at six weeks post vaccination; both K-strain and F-strain vaccination resulted in significant protection from air sac and tracheal lesions, as well as R-strain colonization (P ≤ 0.05). In Trial 2, commercial layer-type chickens were vaccinated with ts-11 (via eye drop) or K-strain (via aerosol) at 12 weeks of age. At 25 weeks of age these birds were challenged with R-strain via aerosol. The ts-11 and K-strain vaccinated groups both had significantly lower air sac lesion scores and a lower prevalence of ovarian regression after challenge as compared to non-vaccinated chickens (P ≤ 0.05). K-strain vaccination also prevented significant tracheal lesions and R-strain colonization (P ≤ 0.05). K-strain shows great potential as a highly efficacious live MG vaccine in broiler and layer-type chickens for protection of the respiratory and reproductive systems as well as prevention of infection with field strains.     

Studies on the persistence and excretion of egg drop syndrome 1976 virus in chickens

EDS virus strain 127 did not induce clinical signs or gross lesions in susceptible chicks of various age groups and in adult cocks. Virus persistence in various internal organs and the rate of excretion of virus by experimentally-infected chicken declined rapidly with increasing age. Virus 127 was detectable in organs of young chicks up to 5 weeks post-infection and in their faeces up to 2 weeks post-infection. In adult birds virus 127 persisted in tissues for about 3 weeks and was excreted with faeces for only 1 week post-infection. Vaccination of adult cocks with an inactivated vaccine resulted in intermittent shedding of virus only within the first 4 days post-challenge; thus, in comparison to non-vaccinated chickens, vaccination reduced virus excretion in faeces to a major extent.     

A study of the pathogenesis of egg drop syndrome in laying hens

Conventionally reared and SPF laying hens were infected with egg drop syndrome (EDS) virus and killed at intervals during the first 13 days post inoculation (pi). Tissues were collected and studied using histo-pathological and immunoperoxidase techniques. EDS viral antigen and intranuclear inclusion bodies were detected in the surface epithelium of the nasal cavity of conventional hens 2 to 6 days pi. Low levels of viral antigen were detected in lymphoid tissue throughout the body 2 to 5 days pi and inflammatory lesions and viral antigen were observed in the infundibulum 3 to 5 days pi. Viral replication was first detected at a low level in the pouch shell gland (PSG) 8 days pi with extensive replication occurring in the PSG of hens killed subsequently. There was a good correlation between the presence of viral antigen and the presence of lesions. Viral antigen was never detected in the surface epithelium of the alimentary tract. These findings suggest that following EDS virus infection, viral replication occurs in the nasal epithelium, viraemia occurs and that the pouch shell gland is the main site of EDS virus replication in the laying hen. It is concluded that the reproductive tract is the most important source of virus for lateral spread in the laying hen.     

The effect of a live vaccine on the horizontal transmission of Mycoplasma gallisepticum.

The effect of a live Mycoplasma gallisepticum vaccine on the horizontal transmission of this Mycoplasma species was quantified in an experimental animal transmission model in specific pathogen free White Layers. Two identical trials were performed, each consisting of two experimental groups and one control group. The experimental groups each consisted of 20 birds 21 weeks of age, which were housed following a pair-wise design. One group was vaccinated twice with a commercially available live attenuated M. gallisepticum vaccine, while the other group was not vaccinated. Each pair of the experimental group consisted of a challenged chicken (10(4) colony-forming units intratracheally) and a susceptible in-contact bird. The control group consisted of 10 twice-vaccinated birds housed in pairs and five individually housed non-vaccinated birds. The infection was monitored by serology, culture and quantitative polymerase chain reaction. The vaccine strain and the challenge strain were distinguished by a specific polymerase chain reaction and by random amplified polymorphic DNA analysis. In both experiments, all non-vaccinated challenged chickens and their in-contact 'partners' became infected with M. gallisepticum. In the vaccinated challenged and corresponding in-contact birds, a total of 19 and 13 chickens, respectively, became infected with M. gallisepticum. Analysis of the M. gallisepticum shedding patterns showed a significant effect of vaccination on the shedding levels of the vaccinated in-contact chickens. Moreover, the Cox Proportional Hazard analysis indicated that the rate of M. gallisepticum transmission from challenged to in-contact birds in the vaccinated group was 0.356 times that of the non-vaccinated group. In addition, the overall estimate of R (the average number of secondary cases infected by one typical infectious case) of the vaccinated group (R = 4.3, 95% confidence interval = 1.6 to 49.9) was significantly lower than that of the non-vaccinated group (R = infinity, 95% confidence interval = 9.9 to infinity). However, the overall estimate of R in the vaccinated group still exceeded 1, which indicates that the effect of the vaccination on the horizontal transmission M. gallisepticum is insufficient to stop its spread under these experimental conditions.     

Experimental assessment of the pathogenicity of the Newcastle disease viruses from outbreaks in Great Britain in 1997 for chickens and turkeys,and the protection afforded by vaccination

The Newcastle disease virus isolated from healthy turkeys in outbreak GB 97/6 was used to challenge 4-week-old turkeys and chickens, which were either not vaccinated or had received a single dose of Hitchner B1 live vaccine 14 days earlier, by one of the intramuscular, intranasal or contact routes. Similar experiments were done in 38-day-old turkeys and chickens using virus isolated from severely sick chickens in outbreak GB 97/1. All vaccinated chickens showed low but measurable immune responses 14 days after vaccination, but only three of the turkeys had detectable antibodies. No vaccinated turkey or chicken showed any clinical sign after challenge with either virus. The virus from healthy turkeys in outbreak GB 97/6 induced clinical signs in 12/30 unvaccinated turkeys after challenge and 7/30 died. In unvaccinated chickens, challenge with this virus produced clinical signs in 25/30 birds and 21/30 died. In challenge experiments with the virus from outbreak GB 97/1 in chickens, 3/30 unvaccinated turkeys showed clinical signs and all three subsequently died. In contrast, 30/30 unvaccinated chickens challenged with this virus showed clinical signs and died. Vaccination did not prevent infection and excretion of either challenge virus. However, when compared with unvaccinated birds, vaccination reduced significantly the length of time virus was excreted and the overall proportion of swabs that were positive.     

Experimental evaluation of an inactivated vaccine against chicken anaemia virus.

An assessment of the performance of an inactivated chicken anaemia virus (CAV) vaccine following administration to specific-pathogen-free (SPF) breeder hens before the egg production period is reported. Antibodies to CAV in adult hens and maternally-derived antibodies to CAV in the progeny chicks were assayed by ELISA. Progeny of vaccinated chickens showed sufficient levels of maternally-derived antibody to CAV for up to 3 to 4 weeks of age. Following challenge at 1, 10, 20 and 30 days of age of chicks from CAV-vaccinated and non-vaccinated hens with virulent CAV, haematocrit levels and weights of thymus in vaccinates remained within normal ranges. Chicks derived from non-vaccinated hens developed anaemia and thymus atrophy following challenge. These results support the view that vaccination of breeder flocks with an inactivated vaccine to CAV could be an effective means of control of CAV-induced clinical disease.     

Evaluation of the protection elicited by direct and indirect exposure to live attenuated infectious laryngotracheitis virus vaccines against a recent challenge strain from the United States

In a recent study (Oldoni & García, 2007), some field strains of infectious laryngotracheitis viruses (ILTV) were characterized as genotypically different (group VI) from ILT vaccine strains. The objective of this study was to evaluate the protection elicited by one chicken embryo origin (CEO) and one tissue culture origin (TCO) vaccine against a field isolate from group VI after direct and indirect exposure to ILTV live attenuated vaccines. In phase 1 of the experiment, non-vaccinated chickens were placed into contact with the eye drop vaccinates for a period of four weeks after vaccination. Transmission of the vaccine virus to these in-contact birds was demonstrated by real time PCR and antibody production, although the in-contact birds did not become protected against disease when subsequently challenged in phase 2 of the experiment. This emphasized the importance of uniform vaccination to obtain adequate protection, both to avoid the occurrence of susceptible chickens, and to minimize the potential for reversion to virulence of live-attenuated vaccines. In phase 2, protection against challenge with a group VI field virus was assessed four weeks after vaccination by scoring clinical signs and mortality, and quantifying weight gain. Sentinel birds were added to the groups one day after challenge to assess shedding of challenge virus, using real time PCR and virus isolation, during the period 2 to 12 days post challenge. The results showed that the CEO and TCO eye drop-vaccinated chickens were protected against challenge with the group VI virus, even though it was genetically different from the vaccine strains, and that challenge virus was not transmitted from these protected birds to the sentinels.     

Long-term protection against a virulent field isolate of infectious laryngotracheitis virus induced by inactivated,recombinant, and modified live virus vaccines in commercial layers

Infectious laryngotracheitis (ILT) is an acute respiratory disease of chickens controlled through vaccination with live-modified attenuated vaccines, the chicken embryo origin (CEO) vaccines and the tissue-culture origin (TCO) vaccines. Recently, novel recombinant vaccines have been developed using herpesvirus of turkey (HVT) and fowl pox virus (FPV) as vectors to express ILTV immunogens for protection against ILT. The objective of this study was to assess the protection efficacy against ILT induced by recombinants, live-modified attenuated, and inactivated virus vaccines when administered alone or in combination. Commercial layer pullets were vaccinated with one or more vaccines and challenged at 35 (35 WCH) or 74 weeks of age (74 WCH). Protection was assessed by scoring clinical signs; and by determining the challenge viral load in the trachea at five days post-challenge. The FPV-LT vaccinated birds were not protected when challenged at 35 weeks; the HVT-LT and TCO vaccines in combination provided protection similar to that observed in chickens vaccinated with either HVT-LT or TCO vaccines when challenged at 35 weeks, whereas protection induced by vaccination with HVT-LT followed by TCO was superior in the 74 WCH group compared with the 35 WCH group. Birds given the inactivated ILT vaccine had fewer clinical signs and/or lower viral replication at 74 WCH when combined with TCO or HVT-LT, but not when given alone. Finally, the CEO-vaccinated birds had top protection as indicated by reduction of clinical signs and viral replication when challenged at 35 weeks (74 weeks not done). These results suggest that certain vaccine combinations may be successful to produce long-term protection up to 74 weeks of age against ILT.     

Protection following simultaneous vaccination with three or four different attenuated live vaccine types against infectious bronchitis virus

ABSTRACT

Two or more different live attenuated infectious bronchitis virus (IBV) vaccine types are often given to broilers to induce homologous protection as well as to broaden protection against other IBV types in the field. However, the ability of broilers to respond to three or four different antigenic types of IBV vaccine has not been examined experimentally. In this study, we vaccinated one-day-old broiler chicks by eyedrop with three or four different IBV vaccine types simultaneously. The presence and relative amount of each vaccine was examined in all of the birds by IBV type-specific real-time RT–PCR at 5 days post-vaccination and each vaccine was detected in all of the birds given that vaccine. The birds were challenged at 28 days of age and protection was measured by clinical signs, virus detection and by ciliostasis. Birds vaccinated with three different IBV types (Ark, Mass and GA98) were protected against challenge with each of those IBV types and were partially protected against challenge with the GA08 virus. Birds vaccinated with four different IBV types (Ark, Mass, GA98 and GA08) were protected against challenge with each of those IBV types with the exception of Mass challenged birds which clearly had 3/11 birds not protected based on individual ciliostasis scores, but had an average ciliostasis score of >50% which is considered protected. The results are important for the control of IBV because they indicate that simultaneous vaccination with up to four different IBV vaccine types can provide adequate protection against challenge for each type.     

The effect of vaccination with a bacterin on the horizontal transmission of Mycoplasma gallisepticum.

The effect of an inactivated vaccine on the horizontal transmission of Mycoplasma gallisepticum was quantified in a transmission model. Twenty non-vaccinated and 20 vaccinated 23-week-old specific pathogen free hens were housed in pairs, while five individually housed hens acted as a negative control group. Each pair consisted of a challenged chicken (10(4) colony forming units intratracheally) and a non-challenged susceptible contact bird. Infection was monitored by serology, quantitative polymerase chain reaction and culture. All non-vaccinated and vaccinated in-contact chickens became infected with M. gallisepticum. The 95% confidence interval of the reproduction ratio, R (a measure of transmission defined as the average number of secondary cases caused by one infectious individual) was 4.48 to infinity in both groups. However, the logarithm of the area under the curve in the vaccinated group was 0.51 lower (P = 0.02) than in the non-vaccinated group, indicating that there was an effect of vaccination on the levels of potential shedding of M. gallisepticum. Nevertheless, the results of this study indicate that the use of an inactivated M. gallisepticum vaccine will not reduce the horizontal transmission of M. gallisepticum between laying hens.