Zataria multiflora essential oil reduces replication rate of avian influenza virus (H9N2 subtype) in challenged broiler chicks.

1. The effect of Zataria multiflora essential oil on replication rate of the H9N2 virus in target organs was determined by real-time PCR. One-day-old broiler chicks were randomly divided into six groups and were challenged with H9N2 influenza. Two groups received either 20 or 40 µl/kg body weight/day Zataria multiflora essential oils (ZM) seven days before the challenge while two other groups received the essential oil at the same dosage but after H9N2 challenge. One group received 4 mg/kg body weight/day of the anti-viral compound amantadine after challenge and the last group received no treatment and served as the control. 2. Groups that received the ZM, before or after H9N2 challenge, and the amantadine treated group showed reduced viral replication in the respiratory and gastrointestinal tracts compared to the control. Supplementation with ZM improved weight gain and FCR in broilers in comparison with the control. 3. The results showed that ZM had a positive effect on reducing viral replication in both the intestine and trachea of H9N2 influenza infected broiler chickens, that led to milder clinical symptoms and better performance.

Sequencing and In Silico Multi-aspect Analysis of S1 Glycoprotein in 793/B Serotype of Infectious Bronchitis Virus Isolated From Iran in 2003 and 2011.

Infectious bronchitis (IB) is an acute, highly contagious, and economically important viral disease of chickens. The S1 subunit from Spike (S) protein plays the major role in protective immunity and is involved in the host-virus interactions, as well as infectious bronchitis virus (IBV) serotyping. Aim of the present study was multi-aspect analysis of the molecular and immunological features of 5' part belonging to the S1 glycoprotein sequence of Iranian 793/B IBV strain isolates. This might ideally help in characterization, prevention, and vaccine development. The tissue samples were prepared, followed by virus isolation, reverse transcription polymerase chain reaction and restriction fragment length polymorphism analysis. In addition, sequencing and registration of the sequences in the National Center for Biotechnology Information were performed. Moreover, 12 sequences were retrieved from Fars province, Iran. The next steps included evaluation of conservation/variability along the sequences, phylogenetic analysis, estimation of the average evolutionary divergence over all the sequence pairs, predicting the phosphorylation/N-glycosylation/palmitoylation sites, and the final analysis of antigenicity. The findings of alignment, entropy plot, and pairwise similarity analysis revealed 17 hypervariable regions. The isolates belonging to Tehran were clustered in phylogenetic tree, and the most similar isolates to them were ADW11182 and ADW11183. Location of some of the N-glycosylation/phosphorylation/palmitoylation points indicated that these sites were conserved among the isolates. Furthermore, the frequency of epitopes and their scores reflect the high immunogenicity of S1 protein in 793/B serotype. Analysis of the primary and secondary structures demonstrated that their parameters had variable values and were different regarding the number and location of α-helix, β-strand, and coils. According to our findings, the Iranian isolates of 793/B serotype change their molecular characteristics during time and in different geographical regions. These alterations might account for failure in prevention programs and differences in virulence and pathogenicity.

Sero-survey of Avian Influenza in backyard poultry and wild bird species in Iran-2014.

In almost all villages in Iran backyard birds, especially chickens, are kept for egg and meat production. AI H9N2 subtype is endemic in Iran. Therefore, estimation of AI prevalence among these birds is important to determine the risk of transmission of infection to commercial farms. The aim of this study was to estimate subclinical infections or previous exposure to H5, H7, and H9 subtypes and to identify potentially important determinants of prevalence of this infectious at premises level in backyard poultry, bird gardens, zoos, and wild bird markets in Iran. A survey was conducted using a cross-sectional design throughout the entire country. A total of 329 villages, seven bird gardens, three zoos and five wild bird markets were included. In each village four families that kept birds were included in the collection of biological samples and background information. The Enzyme-Linked Immunosorbent Assay (ELISA) was used as the screening test and all ELISA-positive samples were examined with the HI test to differentiate H5, H7, and H9. Among the bird gardens, eight of 15 premises (53.3%) were positive in both the ELISA test and HI for H9N2. Testing of samples collected in the villages revealed that 296 out of 329 villages (90%) had positive ELISA tests and also HI tests for H9. The HI-H9 mean titers in positive units were significantly higher than negative units (P<.001). This study revealed no significant statistical differences between risk variables in seropositive and seronegative bird gardens in the case of H9 (P>.05). The results of this study showed that among the risk variables, mountainous area was a protective factor and lack of hygienic disposal of dead birds was a risk factor for AI; this was also observed in rural poultry. The high sero-prevalence of influenza H9N2 in rural domestic poultry indicates that the disease is endemic. It is necessary to include backyard poultry in any surveillance system and control strategy due to the existence of AIV in backyard poultry and the possibility of transmission of infection to commercial poultry farms. Implementation of an AI surveillance program and biosecurity measures can be useful to control this infection and prevent AI from spreading to commercial farms. Furthermore in Iran there is no program for destruction of birds infected with the H9N2, so an effective vaccination program with regard to issues such as acceptability and cost-benefit must play an important role in reducing infections in backyard poultry.

Identification by monoclonal antibodies of serotype D strains of Pasteurella multocida representing various geographic origins and host species.

Two outer-membrane proteins (OMPs) of Pasteurella multocida serotype D, designated H and W, possess potentially important serotype D-specific antigens. Antigenicity as well as toxigenicity of 55 strains of P. multocida representing various serotypes, geographic origins and host species were studied by SDS-PAGE, enzyme-linked immunosorbent assay (ELISA), immunoblot and polymerase chain reaction (PCR) assays. Based on the electrophoretic mobility of protein H, different OMP patterns were observed within different capsular serotypes. Three monoclonal antibodies (MAbs) designated MT1, MT2 and MT3 were produced against H and W proteins of P. multocida in BALB/c mice. MAbs MT2 and MT3 reacted with two distinct epitopes on W protein of serotype D in competitive ELISA. MAb MT1 reacted with all serotype D-I strains but not with D-II strains, whereas MAb MT2 reacted with both serotype D-I and D-II strains in dot-ELISA and immunoblot assay. MAb MT3 reacted with all P. multocida strains belonging to different capsular serotypes in dot-ELISA. None of the MAbs reacted with other gram-negative bacteria tested, indicating that protein H has a serotype D-I specific epitope and protein W has both serotype and species-specific epitopes. PCR assay was used to identify toxigenic strains of P. multocida; 92% of P. multocida strains possess both toxA gene and MAb MT2 reacting epitope, suggesting a strong association between MAb MT2 reacting epitopes and toxA gene. Rapid dot-ELISA with MAb was found to be specific, sensitive and easy to perform and thus suitable for routine serotyping of P. multocida serotype D strains which might be potentially pathogenic.

Detection of 793/B serotype of infectious bronchitis virus in tissue sample by indirect immunoperoxidase assay.

The indirect immunoperoxidase (IIP) assay was compared with the reverse transcription-polymerase chain reaction (RT-PCR) for detection of 793/B serotype of infectious bronchitis virus in tissues samples collected from experimentally infected chickens. This technique was optimized in specific pathogen-free (SPF)-embryonated chicken eggs and broiler chickens inoculated with the Iranian IR/773/2001 strain of 793/B serotype The trachea, lung, kidney, and cecal tonsil tissue samples from experimentally infected chicken embryos and chickens were collected in order to prepare tissue sections in IIP assay and to detect in RT-PCR. The sensitivity and specificity values of IIP assay were, respectively, 83 and 84 %, and the positive and negative prediction values were 71 and 91 % when compared with RT-PCR.

Role of outer membrane protein H (OmpH)- and OmpA-specific monoclonal antibodies from hybridoma tumors in protection of mice against Pasteurella multocida.

Two major outer membrane proteins of Pasteurella multocida, designated OmpH and OmpA, were characterized and shown to be related to the families of porin and heat-modifiable proteins, respectively. The backpack hybridoma tumor system in BALB/c mice was used to continuously deliver immunoglobulin G2b (IgG2b) monoclonal antibodies (MAbs) specific for OmpH (MAb MT1) and OmpA (MAb MT4.1). MAbs were detected in serum and peritoneal lavage samples of mice bearing hybridoma tumors by an enzyme-linked immunosorbent assay and an immunoblot assay. Highly significant protection was observed in mice bearing MT1 hybridoma tumors against both intraperitoneal and intranasal challenge infections with homologous nontoxigenic P. multocida strains possessing MAb MT1-reacting epitopes, whereas the mice bearing MT4.1 hybridoma tumors were not protected. The numbers of P. multocida organisms in the lungs of mice bearing MT1 hybridoma tumors were significantly less than those in lungs of mice bearing MT4.1 hybridoma tumors at 48 h postchallenge. These results indicate that the OmpH-specific MAb inhibited proliferation of P. multocida in the lungs. MAb MT1 was unable to kill P. multocida in vitro in the presence of complement. However, an enhanced phagocytosis by polymorphonuclear cells (PMNs) was observed in mice bearing MT1 hybridoma tumors. P. multocida induced a more extensive and rapid influx of PMNs into the peritoneal cavity of mice bearing MT1 hybridoma tumors than of mice bearing MT4.1 hybridoma tumors. The results of this study demonstrate for the first time that IgG MAbs against OmpH of P. multocida are involved in the protection of mice against lethal challenge infection by means of opsonization and inhibition of proliferation of P. multocida as a result of increased influx of PMNs into the infection site.

The 32 kDa major outer-membrane protein of Pasteurella multocida capsular serotype D.

The major outer-membrane protein (MOMP) of Pasteurella multocida serotype D strain P210, with an apparent molecular mass of 32 kDa, was purified and characterized. The purification method involved selective extraction of MOMP with N-lauroylsarcosine and SDS, followed by immunoaffinity chromatography using a murine monoclonal antibody (mAb). The N-terminal sequence and amino acid composition of the MOMP showed considerable similarity to other Gram-negative bacterial porins, notably to the 37 kDa MOMP (porin H) of P. multocida. Immunoelectron microscopy and colony blotting assays were used to demonstrate the surface localization of the 32 kDa MOMP on bacterial cells. The colony blotting assay provided a simple, sensitive and rapid screening method for visualizing accessibility of the antibody on the cells. In a Western blot assay, murine polyclonal hyperimmune serum against the purified 32 kDa MOMP recognized both serotype B and D strains bearing either a 32 kDa or a 37 kDa MOMP, whereas the mAb recognized only serotype D strains bearing a 32 kDa but not a 37 kDa MOMP. The present data indicate that the 32 kDa MOMPs of P. multocida are antigenically heterogeneous and possess both specific and cross-reacting epitopes. Detection of type-specific epitopes on the 32 kDa MOMP using an mAb may have potential implications regarding the feasibility of developing a serotyping system for P. multocida.