Prediction of Pasteurella multocida serotypes based on whole genomic sequences.

The serotypes of Pasteurella multocida were predicted based on whole genomic sequences (WGS) with specific genes of the capsular and liposaccharide (LPS) outer core polysaccharide regions as targets. A total of 56 strains were whole genomic sequenced and in addition all assembled genomes from NCBI were included for comparison. BIGSdb (Bacterial Isolate Genome Sequence Database) was installed on a Linux server and targets for capsular types A, B, D, E and F were defined as gene sequences of hyaD, bcbD, dcbF, ecbJ and fcbD, respectively and targets for LPS groups 1, 2, 3, 4, 5, 6, 7 and 8 were defined as gene sequences of pcgB, nctA, gatF, latB, rmlA, nctB, ppgB and natG, respectively. The serotypes of P. multocida were predicted from WGS by designating the capsular type and LPS group as well as subtype alleles to isolates. Comparisons between WGS predictions of capsular types and classical phenotypic typing showed correspondence in 87 % of cases whereas comparisons of WGS predictions of LPS groups to phenotypic typing corresponded for 82 % of the strains. In total 93 % and 94 % of the strains available with WGS could be capsular and LPS group typed, respectively. The server is free to access from https://ivsmlst.sund.ku.dk.

Prediction of Mannheimia haemolytica serotypes based on whole genomic sequences.

The aim of the investigation was to predict the serotypes of M. haemolytica based on whole genomic sequences with the capsular gene region as target. A total of 22 strains selected to have been serotyped and to represent all serotypes were investigated by whole genomic sequencing. The BIGSdb (Bacterial Isolate Genome Sequence Database) was downloaded and installed on a Linux server. Here the sequence database was setup with unique loci at serotype level. The server allows serotypes of M. haemolytica to be predicted from whole genomic sequences and the service is available to the public for free from https://ivsmlst.sund.root.ku.dk.

Mucosal application of cationic poly(D,L-lactide-co-glycolide) microparticles as carriers of DNA vaccine and adjuvants to protect chickens against infectious bursal disease.

Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens. The virus protein (VP) 2 induces neutralizing antibodies, which protect chickens against the disease. The aim of this study was to develop a cationic poly(d,l-lactide-co-glycolide) (PLGA) microparticle (MP) based IBDV-VP2 DNA vaccine (MP-IBDV-DNA) for chickens to be delivered orally and by eye drop route. The tested IBDV-VP2 DNA vaccines were immunogenic for specific-pathogen-free chickens and induced an antibody response after intramuscular application. Co-inoculation with a plasmid encoding chicken IL-2 (chIL-2) or CpG-ODN did not significantly improve protection against IBDV challenge. However, the application of a MP-IBDV-DNA vaccine alone or in combination with a delayed oral and eye drop application of cationic MP loaded with CpG-ODN or chIL-2 improved protection against challenge. The MP-IBDV-DNA-vaccinated chickens showed less pathological and histopathological bursal lesions, a reduced IBDV antigen load as well as T-cell influx into the bursa of Fabricius (BF) compared to the other groups (p<0.05). The addition of chIL-2 loaded MP improved challenge virus clearance from the BF as demonstrated by lower neutralizing antibody titers and reduced IL-4 and IFN-α mRNA expression in the bursa at 7 days postchallenge compared to the other challenged groups. Overall, the efficacy of the IBDV-DNA vaccine was improved by adsorption of the DNA vaccine onto cationic PLGA-MP, which also allowed mucosal application of the DNA vaccine.

Reproducibility of swollen sinuses in broilers by experimental infection with avian metapneumovirus subtypes A and B of turkey origin and their comparative pathogenesis.

Swollen head syndrome (SHS) associated with avian metapneumovirus (aMPV) subtype A or subtype B in broilers and broiler breeders has been reported worldwide. Data about pathogenesis of aMPV subtypes A and B in broilers are scarce. It has been difficult to reproduce swollen sinuses in chickens with aMPV under experimental conditions. In the field, SHS in broilers is suspected to be induced by combined infections with different respiratory pathogens. The objectives of the present study were to compare the pathogenesis of subtypes A and B aMPV in commercial broilers and to investigate the reproducibility of clinical disease. In two repeat experiments, commercial broilers free of aMPV maternal antibodies were inoculated with aMPV subtypes A and B of turkey origin. The clinical signs such as depression, coughing, nasal exudates, and frothy eyes appeared at 4 days post inoculation, followed by swelling of periorbital sinuses at 5 days post inoculation. Higher numbers of broilers showed clinical signs in subtype-B-inoculated compared with subtype-A-inoculated groups. Seroconversion to aMPV was detectable from 10 to 11 days post inoculation. The appearance of serum aMPV enzyme-linked immunosorbent assay antibodies and the clearance of the aMPV genome coincided. Subtype B aMPV showed a broader tissue distribution and longer persistence than subtype A. Histopathological changes were observed in the respiratory tract tissues of aMPV-inoculated broilers, and also in paraocular glands, such as the Harderian and lachrymal glands. Overall, our study shows that representative strains of both aMPV turkey isolates induced lesions in the respiratory tract, accompanied by swelling of infraorbital sinuses, indicating the role of aMPV as a primary pathogen for broilers.

A genetically engineered prime-boost vaccination strategy for oculonasal delivery with poly(D,L-lactic-co-glycolic acid) microparticles against infection of turkeys with avian Metapneumovirus.

In this study we demonstrated the use of an oculonasally delivered poly(D,L-lactic-co-glycolic acid) microparticle (PLGA-MP)-based and genetically engineered vaccination strategy in the avian system. An avian Metapneumovirus (aMPV) fusion (F) protein-encoding plasmid vaccine and the corresponding recombinant protein vaccine were produced and bound to or encapsulated by PLGA-MP, respectively. The PLGA-MP as the controlled release system was shown in vitro to not induce any cytopathic effects and to efficiently deliver the F protein-based aMPV-vaccines to avian cells for further processing. Vaccination of turkeys was carried out by priming with an MP-bound F protein-encoding plasmid vaccine and a booster-vaccination with an MP-encapsulated recombinant F protein. Besides the prime-boost F-specific vaccinated birds, negative control birds inoculated with a mock-MP prime-boost regimen as well as non-vaccinated birds and live vaccinated positive control birds were included in the study. The MP-based immunization of turkeys via the oculonasal route induced systemic humoral immune reactions as well as local and systemic cellular immune reactions, and had no adverse effects on the upper respiratory tract. The F protein-specific prime-boost strategy induced partial protection. After challenge the F protein-specific MP-vaccinated birds showed less clinical signs and histopathological lesions than control birds of mock MP-vaccinated and non-vaccinated groups did. The vaccination improved viral clearance and induced accumulation of local and systemic CD4+ T cells when compared to the mock MP-vaccination. It also induced systemic aMPV-neutralizing antibodies. The comparison of mock- and F protein-specific MP-vaccinated birds to non-vaccinated control birds suggests that aMPV-specific effects as well as adjuvant effects mediated by MP may have contributed to the overall protective effect.

Induction of local and systemic immune reactions following infection of turkeys with avian Metapneumovirus (aMPV) subtypes A and B.

Most of the studies regarding the immunopathogenesis of avian Metapneumovirus (aMPV) have been done with subtype C of aMPV. Not much is known about the immunopathogenesis of aMPV subtypes A and B in turkeys. Specifically, local immune reactions have not been investigated yet. We conducted two experiments in commercial turkeys. We investigated local and systemic humoral and cell mediated immune reactions following infection with an attenuated vaccine strain of aMPV subtype B (Experiment I) and virulent strains of aMPV subtypes A and B (Experiment II). Turkeys infected with virulent aMPV strains developed mild respiratory signs while birds inoculated with the attenuated aMPV did not show any clinical signs. Virus neutralizing antibodies were detected locally in tracheal washes and systemically in serum as soon as 5-7 days post aMPV infection (PI) independent of the strain used. Virus neutralizing antibody titres peaked at 7 days PI and then antibody levels declined. The peak of serum ELISA antibody production varied between infected groups and ranged from 14 and 28 days PI. All aMPV strains induced an increase in the percentage of CD4+ T cell populations in spleen and Harderian gland at days 7 or 14 PI. Furthermore, as shown in Experiment I, infection with the attenuated aMPV-B strain stimulated spleen leukocytes to release significantly higher levels of interferons (IFNs), interleukin-6 and nitric oxide in ex vivo culture in comparison to virus-free controls up to 7 days PI (P<0.05). As detected by quantitative real time RT-PCR in Experiment II, infection with virulent aMPV induced an increased IFNgamma expression in the Harderian gland in comparison to virus-free controls. IFNgamma expression in the spleen varied between aMPV strains and days PI. Overall, our study demonstrates that aMPV subtypes A and B infection induced humoral and cell mediated immune reactions comparable to subtype C infections. We observed only temporary stimulation of serum virus neutralizing antibodies and of most of the local immune reactions independent of the aMPV strain used. The temporary character of immune reactions may explain the short duration of protection against challenge following aMPV vaccination in the field.