The role of the major viral pathogens in a respiratory disease outbreak of broiler flocks in Eastern Iran.

Background: Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and avian influenza virus (AIV) H9N2 are major viral pathogens in broiler respiratory disease. Aims: Following a respiratory disease outbreak and economic losses in eastern Iran 2020-2021, we investigated the role of major viral pathogens and the implemented vaccination programs. Methods: Thirty-six respiratory disease affected broiler flocks in South Khorasan province were sampled, molecularly tested, and coinfections were investigated. The vaccination programs were obtained and the detected IBV were genotyped. Results: IBV, virulent NDV, and AIV H9N2 were detected in twenty-five, seven, and seven flocks, respectively. IBV+AIV, IBV+NDV, and NDV+AIV coinfections were respectively detected in six, five, and one flocks. Most IBV infected flocks (84%) had been immunized with a live IBV-Mass vaccine. All NDV infected flocks and 14.2% of AIV infected flocks had been vaccinated. IBV genotyping showed a high prevalence of variant 2 (83.3%), followed by Mass-type (12.5%), and Q1-type (4.2%). Variant 2 IB viruses were widely distributed in the province and half of them were mostly similar to the ones that had been detected in northern neighboring province, Khorasan Razavi. Conclusion: Single infection with variant 2 IBV was a major cause of the respiratory disease outbreak in which use of the Mass vaccine was probably not effective. The high coverage and multiple doses of vaccination against Newcastle disease possibly had reduced the prevalence of NDV. Considering the regional origin of IBV strains, strong biosecurity measures should be implemented and vaccination programs using appropriate vaccine strains should be used.

Improvement in the Growth and α-toxin Production of Clostridium septicum by Magnesium Sulfate.

Clostridium septicum, the anaerobic toxigenic bacterium is the agent that causes dangerous disease in man and animals. There is a lethal toxin of the bacterium namely alpha toxin. The ɑ-toxin has hemolytic, necrotic and lethal activities. Today, Razi Vaccine and Serum Research Institute of Iran produced the C. septicum vaccine in the form of bacterin/toxoid. Because of some problems, the vaccine needs to improve on an industrial scale. The study is going to find an appropriate supplement to improve growth and ɑ-toxin production. Three strains of C. septicum (vaccine, NH1 and NH8 strains) were cultured in the basic vaccine media. Magnesium sulfate, Copper, Ferrous, yeast extract, and trace elements plus vitamins' solution were added to the basic vaccine media in different cultures. The effect of the ingredients on the growth was measured by a spectrophotometer and the α-toxin secretion was assayed by hemolysin test. Growth of the bacterium and α-toxin secretion were increased by Magnesium (80 mg/l) in NH8 and vaccine strains significantly. The black precipitate was difficult to dissolve in magnesium media that must be solved. Trace elements plus vitamins solution mildly influence on NH1strain growth and toxin secretion. Other supplements (Cu, Fe, yeast extract) were not showen any significant changes in the growth and α-toxin production of C. septicum. Overflowing peptone (4%) in the vaccine media, fixes essentials of proteolysis activity, allows the sufficient growth and toxin production without Cu, Fe, and yeast extract. Due to essentially of Mg for growth, extra magnesium was added for improvement of media culture. The study suggests for Magnesium addition in the C. septicum vaccine media during production procedure after precipitation solving problem.

Production and characterization of egg yolk antibody (IgY) against recombinant VP8-S2 antigen.

Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. VP8 subunit of rotavirus is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Studies showed that immunoglobulin of egg yolk (IgY) from immunized hens has been identified to be a convenient source for specific antibodies for using in immunotherapy and immunodiagnostic to limit the infections. In this study, chimeric VP8-S2 gene was designed using by computational techniques. The chimeric VP8-S2 gene was cloned and sub-cloned into pGH and pET32a (+) vectors. Then, recombinant pET32a-VP8-S2 vector was transferred into E. coli BL21 CodonPlus (DE3). The expressed protein was purified by Ni-NTA chromatography column. Hens were immunized with the purified VP8-S2 protein three times. IgY was purified from egg yolks using polyethylene glycol precipitation method. Activity and specificity of anti-VP8-S2 IgY were detected by dot-blotting, Western-blotting and indirect ELISA. We obtained anti-VP8-S2 IgY by immunizing hens with the recombinant VP8-S2 protein. The anti-VP8-S2 IgY was showed to bind specifically to the chimeric VP8-S2 protein by dot-blotting, Western-blotting analyses and indirect ELISA. The result of this study indicated that such construction can be useful to investigate as candidates for development of detection methods for simultaneous diagnosis of both infections. Specific IgY against the recombinant VP8-S2 could be recommended as a candidate for passive immunization against bovine rotavirus and bovine coronavirus.

Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system.

The impending influenza virus pandemic requires global vaccination to prevent large-scale mortality and morbidity, but traditional influenza virus vaccine production is too slow for rapid responses. In this study, bacterial system has been developed for expression and purification of properly folded HA1 antigen as a rapid response to emerging pandemic strains. Here, a recombinant H5N1 (A/Indonesia/05/05) hemagglutinin globular domain, the synthesized HA1 (1-320 amino acids), was amplified and cloned into pET-28a bacterial expression vector. Then, his-tagged HA1 protein was expressed in Escherichia coli BL21 under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA chromatography. Migration size of protein was detected at 40 KDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight. Since most antigenic sites are in the HA1 domain of HA, using this domain of influenza virus as antigen is of great importance in vaccine development. The ability of the antibody stimulation against HA1 expressed in bacterial cells is also examined using enzyme-linked immunosorbent assay (ELISA) analysis. Upon immunization of rabbits, oligomeric HA1 elicited potent neutralizing antibodies and high levels of serum antibody binding to HA1. Our findings suggest that HA1-based vaccines can be produced efficiently in bacterial systems and can be easily upscaled in response to a pandemic influenza virus threat.

Molecular investigation and cultivation of camelpox virus in Iran.

Camelpox virus (genus Orthopoxvirus, family Poxviridae) is the etiologic agent of camel pox. The clinical manifestations of this virus range from inapparent infection to mild, moderate and, less commonly, severe systemic infection and death. Following an outbreak of camelpox, samples that were collected from camel flocks suspected to have camelpox in Qom Province in central Iran and Khash city, Sistan and Baluchestan Province and South Khorasan Province in eastern Iran were sent to Razi Vaccine and Serum Research Institute in Mashhad. DNA extraction was performed primarily by the phenol-chloroform method, and PCR was carried out using a Bioneer kit. Using the primer pair 5'-AAT-ACA-AGG-AGG-ATC-T-3' and 5'-CTT-AAC-TTT-TTC-TTT-CTC-3', the gene sequence encoding the A-type inclusion protein (ATIP) was amplified. The size of the PCR product, specific for camelpox virus, was 881 bp. The PCR product was purified, and to confirm its sequence, it was sent to the reference laboratory. The sequence was subjected to a BLAST search and then phylogenetically analyzed using CLC software. The results showed that all samples were nearly 100 % identical to each other and to strains CMS and M-96. These isolates also had 99 % and 95 % similarity to the CP-1 strain and isolate FIN/T2000, respectively. In Vero cell culture, inoculation with this virus caused a cytopathic effect (CPE), which appeared 2-5 days post-inoculation. Characteristic CPE showing foci of rounded cells, ballooning, giant-cell formation and syncytia with degenerative changes appeared.

A survey on the frequency of foot-and-mouth disease virus carriers in cattle in north-east of Iran by RT-PCR: implications for revising disease control strategy.

Foot-and-mouth disease (FMD) is endemic in Iran. It is essential to timely evaluate the current disease control programme in Iran. Here, we report the frequency of FMD virus (FMDV) carrier state in cattle slaughtered in Mashhad abattoir, Mashhad, Khorasan Razavi, north-east of Iran, which contains long common borders with Afghanistan and Turkmenistan. Soft palate samples were collected immediately after slaughter for the detection of FMDV by RT-PCR. The results show that 37.7% of cattle (96 of 255) were carriers of the virus. Among positive samples (96), 58 (60.4%) belonged to serotype O. No evidence was detected for the presence of Asia 1 and A serotypes. Nucleotide sequencing and phylogenic dendogram showed close similarity and common lineage between our samples and viruses isolated in Pakistan. With an approximate more than 80% of cattle population vaccination coverage such a high rate of carrier state may show an extensive FMDV exposure. Therefore, limiting control programmes to timely prophylactic vaccination may be insufficient. This is also true when meat market instabilities act as a temptation to import livestock, legally or illegally, through the eastern frontiers. It is recommended to change the current prophylactic vaccination strategy to a well-developed regional control programme, with close monitoring of animal movement through eastern frontiers, supported by government commitment and educational programmes. Timely estimation of the frequency of carrier state both in cattle and small ruminants is also advocated as a gauge to monitor the virus status in the region.