Vaccinating chickens against avian influenza with fowlpox recombinants expressing the H7 haemagglutinin

OBJECTIVE: To evaluate the vaccine efficacy of a fowlpox virus recombinant expressing the H7 haemagglutinin of avian influenza virus in poultry. PROCEDURE: Specific-pathogen-free poultry were vaccinated with fowlpox recombinants expressing H7 or H1 haemagglutinins of influenza virus. Chickens were vaccinated at 2 or 7 days of age and challenged with virulent Australian avian influenza virus at 10 and 21 days later, respectively. Morbidity and mortality, body weight change and the development of immune responses to influenza haemagglutinin and nucleoprotein were recorded. RESULTS: Vaccination of poultry with fowlpox H7 avian influenza virus recombinants induced protective immune responses. All chickens vaccinated at 7 days of age and challenged 21 days later were protected from death. Few clinical signs of infection developed. In contrast, unvaccinated or chickens vaccinated with a non-recombinant fowlpox or a fowlpox expressing the H1 haemagglutinin of human influenza were highly susceptible to avian influenza. All those chickens died within 72 h of challenge. In younger chickens, vaccinated at 2 days of age and challenged 10 days later the protection was lower with 80% of chickens protected from death. Chickens surviving vaccination and challenge had high antibody responses to haemagglutinin and primary antibody responses to nucleoprotein suggesting that although vaccination protected substantially against disease it failed to completely prevent replication of the challenge avian influenza virus. CONCLUSION: Vaccination of chickens with fowlpox virus expressing the avian influenza H7 haemagglutinin provided good protection against experimental challenge with virulent avian influenza of H7 type. Although eradication will remain the method of first choice for control of avian influenza, in the circumstances of a continuing and widespread outbreak the availability of vaccines based upon fowlpox recombinants provides an additional method for disease control.     

Immunogenicity, pathogenicity, and transmissibility of a recombinant vaccinia virus in calves

Experiments concerned with the immunogenicity, pathogenicity, and transmissibility of a recombinant vaccinia:Sindbis virus were conducted. The WR strain of the recombinant vaccinia:Sindis virus was found to be infective for calves and mildly pathogenic, resulting in local tissue reaction. It was not transmissible to other calves. Also, it was found to be immunogenic when inoculated intradermally into calves, and antibody was produced against the parent vector virus (vaccinia) and the Sindbis antigen. Recombinant virus given IV to calves induced no detectable clinical signs, nor did the calves develop neutralizing antibodies. Furthermore, second-passage lesion material containing up to 10(7) tissue culture infective doses of the recombinant virus failed to induce development of lesions or illness in intradermally inoculated calves, and virus could not be recovered from the inoculation sites. In this series of experiments, this vaccinia recombinant given intradermally was immunogenic, mildly pathogenic at the local injection site only, and was not transmissible to contact animals, thus demonstrating the potential efficacy and safety of the WR strain of vaccinia virus when used as a live vector system in cattle.     

Immune responses of swine inoculated with a recombinant fowlpox virus co-expressing P12A and 3C of FMDV and swine IL-18

Two recombinant fowlpox viruses (rFPV-P1 and rFPV-IL18-2AP12A) containing foot-and-mouth disease virus (FMDV) capsid polypeptide, 3C coding regions of O/NY00 were evaluated to determine their abilities to induce humoral and cellular responses in the presence or absence of swine IL-18 as genetic adjuvant. The ability to protect swine against homologous virus challenge was examined. All swine were given booster vaccinations at 21 days after the initial inoculation and were challenged 10 days after the booster vaccination. Control groups were inoculated with wild-type fowlpox virus (wtFPV). All animals vaccinated with rFPV-P12A and rFPV-IL18-P12A developed specific anti-FMDV ELISA antibody and neutralizing antibody and T-lymphocyte proliferation was observed. Cellular immune function was evaluated via examination of IFN-gamma production in swine peripheral blood serum. The results demonstrate the potential viability of a fowlpox virus-based recombinant vaccine in the control and prevention of FMDV infections.     

Mechanisms of infection in the respiratory tract

Related to its potential vulnerability the respiratory tract has a very complex and effective defence apparatus. The interaction between these defence mechanisms and certain characteristics of aetiological agents results in a pattern in which initial infections by these agents tend to occur at specific sites in the tract. Infections in which the primary portal of entry is in the upper respiratory tract include Bordetella bronchiseptica and Haemophilus spp in pigs; Pasteurella spp in cattle, sheep, pigs; Mycoplasma spp in cattle, sheep, pigs and poultry; equine herpesvirus 1 in horses; infectious bovine rhinotracheitis in cattle; parainfluenza 3 in cattle and sheep; infectious laryngo-tracheitis and infectious bronchitis in poultry; feline viral rhinotracheitis and calicivirus in cats; Aujeszky's disease virus and swine influenza in pigs; and equine influenza in horses. Infections in which the primary portal of entry is in the lower respiratory tract include Aspergillus fumigatus in poultry and mammals, respiratory syncytial virus in cattle, distemper virus in dogs and adenovirus in cattle and dogs. A fuller understanding of the interactions between an agent and the host at the point of entry would make it much easier to develop effective vaccines and therapeutic agents.     

Evaluation of the pathogenicity and immunogenicity of vaccinia virus to piglets

The pathogenicity and immunogenicity of vaccinia virus were examined in order to evaluate the possibility of its application as a recombinant viral vaccine in pigs. Following scarification inoculation with vaccinia virus, a mild reddish papulation developed only on the scarified part of the skin. No symptoms of illness such as fever or stunting were noted. Vaccinia virus was recovered in titers from scarified skin 4 and 7 days after inoculation. Control piglets cohabited with inoculated animals remained normal for the whole 5 week observation period. Hemagglutination inhibition and indirect immunofluorescence tests detected antibodies against vaccinia virus in the inoculated piglets, whereas no anti-vaccinia virus antibodies were detected in the contact control animals. Antigen-induced blastogenic tests of peripheral blood lymphocytes from animals, revealed that lymphocytes obtained from inoculated donors 5 weeks after inoculation, had a higher stimulation index (P less than 0.05) than did those from uninoculated piglets. These results suggested that vaccinia virus would be useful as a recombinant viral vector for pigs.     

Canine pedal dermatitis due to percutaneous Uncinaria stenocephala infection

Related to its potential vulnerability the respiratory tract has a very complex and effective defence apparatus. The interaction between these defence mechanisms and certain characteristics of aetiological agents results in a pattern in which initial infections by these agents tend to occur at specific sites in the tract.

Infections in which the primary portal of entry is in the upper respiratory tract include Bordetella bronchiseptica and Haemophilus spp in pigs; Pasteurella spp in cattle, sheep, pigs; Mycoplasma spp in cattle, sheep, pigs and poultry; equine herpesvirus 1 in horses; infectious bovine rhinotracheitis in cattle; parainfluenza 3 in cattle and sheep; infectious laryngo-tracheitis and infectious bronchitis in poultry; feline viral rhinotracheitis and calicivirus in cats; Aujeszky' disease virus and swine influenza in pigs; and equine influenza in horses. Infections in which the primary portal of entry is in the lower respiratory tract include Aspergillrrs fumigatus in poultry and mammals, respiratory syncytial virus in cattle, distemper virus in dogs and adenovirus in cattle and dogs. A fuller understanding of the interactions between an agent and the host at the point of entry would make it much easier to develop effective vaccines and therapeutic agents.     

Role of IFN-α during the acute stage of a swine influenza virus infection

Cytokines, especially interferon-alpha (IFN-α) are important in controlling influenza virus infections. To investigate the role of IFN-α in influenza, the swine IFN-α neutralizing monoclonal antibody (Ab) K9 was applied in a swine model of influenza A virus infection. First, the optimal dose and route for administration of the IFN-α neutralizing Abs was determined. Based on those results, the effect of the Abs on a swine influenza virus infection was investigated. Pigs were inoculated intratracheally with 10^6.0 mean egg infectious dose (EID₅₀) A/Swine/Belgium/1/98 (H1N1) virus. At the time of challenge and 18 h later, they were injected intratracheally and intraperitoneally with a high dose of IFN-α neutralizing Abs or control Abs. The animals were euthanized at 0, 24, 30, 48 and 72 h after inoculation. At 24 and 30 h, IFN-α levels in broncho-alveolar lavage fluid of K9 recipient animals were strongly suppressed, and this coincided with reduced IL-6 and IL-12 levels. TNF-α and IL-1 levels were unaffected compared to those in the control Ab treated group. Importantly, the onset and peak of clinical symptoms in IFN-α neutralizing Abs treated animals were delayed by 24 h, simultaneously with the suppression of IFN-α, but there was no obvious effect on virus replication and lung pathology. These results suggest an important role for IFN-α in IL-6 and IL-12 induction and a role of all three cytokines in the symptoms of swine influenza.     

Immune response and protection of cattle and pigs generated by a vaccinia virus recombinant expressing the F protein of rinderpest virus

The immune response of cattle and pigs to a vaccinia recombinant virus containing the fusion (F) protein gene of rinderpest virus was examined. Half the cattle and all the pigs gave humoral response to primary vaccination and all the cattle gave an anamnestic response to a second vaccination 28 days after the primary vaccination. All the cattle after a single or secondary vaccination were completely protected clinically after exposure to a lethal dose of the Saudi 1/81 strain of virus. Prior vaccination with another TK- vaccinia recombinant (VVCAT) suppressed, but did not abrogate, the immune response to the rinderpest F recombinant. The pigs gave a humoral immune response in the absence of any local reaction at the site of vaccination.     

Humoral immune response of BALB/c mice to a vaccinia virus recombinant expressing Brucella abortus GroEL does not correlate with protection against a B. abortus challenge

This work is a part of an ongoing effort to develop vaccinia virus recombinants expressing various Brucella abortus proteins. The B. abortus groEL gene encoding the antigenic heat shock protein GroEL was subcloned into vaccinia virus via homologous recombination and expression confirmed by Western blotting. Female BALB/c mice inoculated with recombinant vaccinia virus/GroEL produced GroEL and vaccinia virus specific antibodies. Mice were challenged 8 weeks post-inoculation with virulent B. abortus strain 2308 and protection measured by the rate of clearance of live Brucella from spleens. Although induction of specific immune response to GroEL and vaccinia virus was demonstrated by the appearance of antibodies in mice, no significant level of protection was demonstrable.     

Recombinant adenovirus expressing GP5 and M fusion proteins of porcine reproductive and respiratory syndrome virus induce both humoral and cell-mediated immune responses in mice

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important contagious agents of swine in the world. PRRSV infection poses a challenge to current vaccination strategies. In this study, three replication-defective adenovirus recombinants were developed as potential vaccine against PRRSV in a mouse model. Three groups of BALB/c mice (24 mice per group) were inoculated subcutaneously twice at 2-week intervals with the recombinants expressing PRRSV GP5 (rAd-GP5), M (rAd-M), and M-GP5 fusion protein (rAd-M-GP5). Two additional groups were injected with wild-type adenovirus (wtAd) or PBS as control. The results showed that the mice inoculated with recombinant adenoviruses developed PRRSV-specific antibodies, cellular immune response by 2 weeks post second inoculation. However, only mice immunized with recombinant adenovirus rAd-M-GP5 developed significantly higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to mice immunized with rAd-M or rAd-GP5 alone. It was also found that mice immunized with rAd-M-GP5 were primed for significant higher levels of anti-PRRSV CTL responses than mice immunized with rAd-M. Mice receiving rAd-GP5 also mounted PRRSV-specific response, but levels were lower. It suggested that the recombinant adenovirus expressing M-GP5 fusion protein might be an attractive candidate vaccine to be tested for preventing PRRSV infection.     

A recombinant pseudorabies virus encoding the HA gene from H3N2 subtype swine influenza virus protects mice from virulent challenge

The hemagglutinin (HA) gene of A/Swine/Inner Mogolian/547/2001 (H3N2) swine influenza virus (SIV) was recombined into the genome of pseudorabies virus (PRV) Bartha-K61 vaccine strain, generating a recombinant PRV expressing the HA gene, designated as rPRV-HA. One group of 15 mice was inoculated intranasally (i.n.) with 10(5.0) PFU of rPRV-HA, and another two control groups of mice (15 mice per group) were mock-inoculated or inoculated with Bartha-K61. Mice inoculated with rPRV-HA developed hemagglutination inhibition antibodies 3 weeks post-inoculation. Twenty-eight days post-inoculation, all mice were challenged i.n. with 10(5.0) TCID50 of A/Swine/Heilongjiang/74/2000 (H3N2). No challenge virus was isolated from vaccinated mice, and mild pathological lesions were observed only in lungs following challenge. The results demonstrate that the recombinant rPRV-HA expressing the HA gene from H3N2 SIV can protect mice from heterologous virulent challenge, and may represent a candidate vaccine against SIV.     

Immune response of pigs inoculated with virulent pseudorabies virus and pigs inoculated with attenuated or inactivated pseudorabies virus vaccine before and after challenge exposure

Pseudorabies virus (PRV) antibodies, detectable by indirect radioimmunoassay (IRIA), serum-virus neutralization test (NT), or microimmunodiffusion test (MIDT) were developed within 8 days after pigs were inoculated with virulent PRV or attenuated PRV vaccine. Indirect radioimmunoassay and NT titers in pigs inoculated with virulent PRV were developed at the same rate, with IRIA titers being higher than NT titers. Pigs inoculated with attenuated or inactivated PRV vaccine developed peak mean prechallenge NT antibody titers of 4 and 1 (reciprocals of serum dilutions), respectively. Pigs inoculated with attenuated PRV vaccine had peak mean prechallenge IRIA antibody titers of 6, whereas pigs inoculated with inactivated PRV vaccine had mean IRIA antibody titers of 64. Challenge exposure of swine inoculated with attenuated or inactivated PRV vaccine elicited quantitatively equivalent responses, as measured by IRIA or NT, which were higher than prechallenge titers. There were no false-positive IRIA, NT, or MIDT results obtained when sera from nonvaccinated, nonchallenge-exposed pigs were tested. It appears that the PRV infection status of a seropositive swine herd could be ascertained by serologically monitoring several representative animals from a herd, using the NT. If 2 or more tests of representative animals at 14-day intervals were done and the mean NT titer was 4 or less, it could be concluded that the herd was vaccinated against, but not infected with, virulent virus.     

Development and validation of a 3ABC indirect ELISA for differentiation of foot-and-mouth disease virus infected from vaccinated animals

Non-structural protein (NSP) 3ABC antibody is considered to be the most reliable indicator of present or past infection with foot-and-mouth disease virus (FMDV) in vaccinated animals. An indirect ELISA was established, using purified His-tagged 3ABC fusion protein as antigen, for detection of the antibody response to FMDV NSP 3ABC in different animal species. The method was validated by simultaneous detection of the early antibody responses to NSP and structural protein (SP) in FMDV Asia 1 infected animals. The performance of the method was also validated by detection of antibody in reference sera from the FMD World Reference Laboratory (WRL) in Pirbright, UK, and comparison with two commercial NSP ELISA kits. The results showed that the antibody response to SP developed more quickly than that to NSP 3ABC in FMDV infected animals. In contact-infected cattle, the antibody response to NSP 3ABC was significantly delayed compared with that to SP antibody. The early antibody responses to SP and NSP 3ABC in FMDV inoculated cattle and contact-infected or inoculated sheep and pigs were generally consistent. In pigs, 3ABC antibody was linked to the presence of clinical signs; however, in sheep, subclinical infection was detected by the development of 3ABC antibodies. Therefore, the antibody responses to 3ABC varied between host species. Eight out of 10 positive serum samples from FMD WRL were tested to be positive at cutoff value of 0.2. The rate of agreement with the ceditest FMDV-NS and the UBI NSP ELISA were 98.05% (302/308) and 93.2% (287/308), respectively. The prevalence of 3ABC antibodies reached 71.4% in some diseased cattle herds. The further work is required to evaluation the performance of this method in different animal species and different field situations.     

BLUETONGUE: THE DISEASE IN CATTLE

Most researchers in South Africa found that although BT virus could be isolated from apparently healthy cattle and from inoculated cattle the virus did not produce overt clinical disease in cattle. However, when epizootics were reported outside Africa, clinical signs were observed in cattle in Israel, Palestine, Syria, Portugal, and Spain. Most natural BT infections in cattle in the United States do not result in overt clinical signs. However, in certain infected herds, approximately 5% of the cattle show from mild to severe disease. Except for severe cases, spontaneous recovery is usual. The clinical diagnosis of BT in cattle is difficult and requires laboratory assistance. Culicoides variipennis can serve as a vector of BT virus from cattle to cattle, cattle to sheep, sheep to cattle, and sheep to sheep. In utero transmission occurs in cattle and can result in abortion, hydraencephaly, congenital deformity, and birth of viraemic calves which may or may not develop BT antibody. Calves inoculated in utero or those born to infected dams may have a persistent viraemia with or without BT antibody. tone such animal has been held in insect-secure quarters and has continued to harbour virus for 3 years. Bluetongue virus was isolated from the semen of experimentally infected bulls. Calves inoculated with BT virus and also given an immuno-suppressant developed marked clinical disease in 8 to 12 days. Bluetongue virus is very closely associated with the erythrocytes of infected cattle, sheep, and goats. Cattle are considered important and relatively long-term virus reservoirs. In attempts to determine the maximum period of viraemia in cattle it is necessary to inoculate washed erythrocytes, rather than whole blood, and to use susceptible sheep as the assay system rather than embryonated chicken eggs.     

New techniques to measure blood cholinesterase activity in domesticated animals.

A macromethod and a semimicromethod were developed to measure erythrocyte acetylcholinesterase activity in cattle, sheep, goats, horses, dogs, and swine, and to measure plasma cholinesterase activity in horses, dogs, and swine. Comparison of the 2 methods with erythrocytes of sheep, cattle, goats, and horses indicated both methods gave similar results. They can be done in a shorter time and are more sensitive than Michel's method. Normal deltapH values per minutes, with standard deviations for blood cholinesterase activity of animals of different ages, sexes, breeds, and species, were: 0.76 +/- 0.12/30; 0.65 +/- 0.10/15; 0.69 +/- 0.19/45; 0.78 +/- 0.11/45; 0.63 +/- 0.11/45; and 0.71 +/- 0.06/25 for sheep, cattle, goats, horses, dogs, and swine erythrocyte acetylcholinesterase, respectively; and 0.66 +/- 0.18/20; 0.67 +/- 0.20/30, and 0.46 +/- 0.05/60 for horses, dogs, and swine plasma cholinesterase, respectively. It was shown that either the chloride or the iodide salt of acetylcholine can be used as the enzyme substrate. tin blood samples stored at 5 C for 24 hours, there was no significant change of the enzymatic activity.     

Zoonotic disease awareness survey of backyard poultry and swine owners in southcentral Pennsylvania

Owners of small backyard poultry and swine operations may be at higher risk of zoonotic diseases due to husbandry inexperience and/or a lack of knowledge. Backyard poultry and swine owners in southcentral Pennsylvania were surveyed regarding their knowledge and attitudes towards zoonotic disease prevention. One hundred and six backyard poultry and/or swine owners completed the survey (74 poultry, 15 swine, 17 both), which included questions on demographics, flock/herd characteristics, recognition of selected zoonotic diseases and clinical signs in animals, and biosecurity practices for visitors and owners. Most responded that they were aware of avian (92.2%) and swine (84.4%) influenza, and were less aware of other zoonotic diseases such as salmonellosis and brucellosis. The majority of backyard poultry and swine owners combined (62.9%) reported allowing visitors freely around their animals and did not require any special precautions. Backyard poultry and swine owners most commonly reported rarely (32.7%) or never (28.9%) wearing work gloves and never (57.1%) wearing nose/mouth coverings, such as a respirator mask, while handling animals or manure. The study findings indicated that veterinarians (61.5%) and the Internet (50.0%) are the main sources where small‐scale farm producers seek animal disease information. Approximately one‐third (34.9%) of the respondents reported receiving seasonal influenza vaccine. The findings of this study will be utilized to provide targeted veterinary and public health education for the prevention of zoonotic diseases in backyard farm animal settings in Pennsylvania.     

Identification and characterization of H2N3 avian influenza virus from backyard poultry and comparison with novel H2N3 swine influenza virus

In early 2007, H2N3 influenza virus was isolated from a duck and a chicken in two separate poultry flocks in Ohio. Since the same subtype influenza virus with hemagglutinin (H) and neuraminidase (N) genes of avian lineage was also identified in a swine herd in Missouri in 2006, the objective of this study was to characterize and compare the genetic, antigenic, and biologic properties of the avian and swine isolates. Avian isolates were low pathogenic by in vivo chicken pathogenicity testing. Sequencing and phylogenetic analyses revealed that all genes of the avian isolates were comprised of avian lineages, whereas the swine isolates contained contemporary swine internal gene segments, demonstrating that the avian H2N3 viruses were not directly derived from the swine virus. Sequence comparisons for the H and N genes demonstrated that the avian isolates were similar but not identical to the swine isolates. Accordingly, the avian and swine isolates were also antigenically related as determined by hemagglutination-inhibition (HI) and virus neutralization assays, suggesting that both avian and swine isolates originated from the same group of H2N3 avian influenza viruses. Although serological surveys using the HI assay on poultry flocks and swine herds in Ohio did not reveal further spread of H2 virus from the index flocks, surveillance is important to ensure the virus is not reintroduced to domestic swine or poultry. Contemporary H2N3 avian influenza viruses appear to be easily adaptable to unnatural hosts such as poultry and swine, raising concern regarding the potential for interspecies transmission of avian viruses to humans.     

Induction of a cross-reactive antibody response to influenza virus M2 antigen in pigs by using a Sendai virus vector

Protecting pigs from simultaneous infection with avian, swine, and human influenza viruses would be an effective strategy to prevent the emergence of reassortants with pandemic potential. M2 protein is a candidate antigen for so-called 'universal vaccines,' which confer cross-protection to different influenza viruses in a strain- and subtype-independent manner. We tested whether a recombinant F gene-deleted Sendai virus vector that contained an M2 gene derived from an H5N1 avian influenza virus (SeV/ΔF/H5N1M2) could induce a cross-reactive antibody response to the extracellular domain of M2 protein (M2e) in pigs. SeV/ΔF/H5N1M2 induced an antibody response to M2e when the vector was inoculated intramuscularly. The antibodies induced by SeV/ΔF/H5N1M2 cross-reacted with M2e derived from different avian, swine, and human influenza viruses. In mice, however, SeV/ΔF/H5N1M2 did not confer cross-protection to challenge with a heterologous H3N2 influenza virus. Our results confirm those of other groups indicating that antibodies to M2e do not mediate protection to influenza viruses in pigs.     

Analysis of the quality of protection induced by a porcine influenza A vaccine to challenge with an H3N2 virus

Antigenic drift of swine influenza A (H3N2) viruses away from the human A/Port Chalmers/1/73 (H3N2) strain, used in current commercial swine influenza vaccines, has been demonstrated in The Netherlands and Belgium. Therefore, replacement of this human strain by a more recent swine H3N2 isolate has to be considered. In this study, the efficacy of a current commercial swine influenza vaccine to protect pigs against a recent Dutch field strain (A/Sw/Oedenrode/96) was assessed. To evaluate the level of protection induced by the vaccine it was compared with the optimal protection induced by a previous homologous infection. Development of fever, virus excretion, and viral transmission to unchallenged group mates were determined to evaluate protection. The vaccine appeared efficacious in the experiment because it was able to prevent fever and virus transmission to the unchallenged group mates. Nevertheless, the protection conferred by the vaccine was sub-optimal because vaccinated pigs excreted influenza virus for a short period of time after challenge, whereas naturally immune pigs appeared completely protected. The immune response was monitored, to investigate why the vaccine conferred a sub-optimal protection. The haemagglutination inhibiting and virus neutralising antibody responses in sera, the nucleoprotein-specific IgM, IgG, and IgA antibody responses in sera and nasal secretions and the influenza-specific lymphoproliferation responses in the blood were studied. Vaccinated pigs developed the same or higher serum haemagglutination inhibiting, virus neutralising, and nucleoprotein-specific IgG antibody titres as infected pigs but lower nasal IgA titres and lymphoproliferation responses. The lower mucosal and cell-mediated immune responses may explain why protection after vaccination was sub-optimal.