Comparison of the Immunogenicity of Four Infectious Bursal Disease Intermediate Vaccines in Commercial Broiler Flocks in Iran: A Field Trial Study.

Infectious bursal disease (IBD) is a highly contagious disease in young chickens worldwide. The major strategy for the prevention and control of IBD virus (IBDV) is vaccination. Therefore, the present study aimed to compare the immunogenicity of four commercially available IBD vaccines on broilers (Ross 308) that were raised in areas with very virulent IBDV infection history. Two commercial broiler farms with four standard poultry houses were selected in Alborz (n=6,250 birds per house) and Khorasan Razavi (n=8,000 birds per house) provinces of Iran. In each farm, the houses were randomly assigned to one of the four IBD intermediate vaccine brands including Dn, Vc, Ch, and Razi. The birds in Alborz were vaccinated against IBDV via drinking water at 18 and 22; and 15 and 21 days of age in Alborz and Khorasan Razavi flocks, respectively. The enzyme-linked immunosorbent assay antibody titers against IBDV were measured in 20 birds per group at 1, 28, 35, and 42 days of age. In addition, production attributes including body weight, feed conversion ratio, mortality, and production index were measured during the research period. According to the findings, the IBD antibody titers were not affected by the vaccine brands at 28, 35, and 42 days of age (P>0.05). Following the second IBD vaccination, an increasing trend in IBD antibody titers was noted in the Razi vaccine as well as other brands at days 35 and 42 compared to the previously recorded titers (P<0.05). Moreover, the production attributes of the flocks receiving various IBDV vaccine brands were not different (P>0.05). Regarding the productivity indices and high immunogenicity levels, the results indicated that the potential of the IBD Razi vaccine was comparable to the other investigated brands of commercial IBD vaccines, and nominated it as an immunogenic candidate vaccine for use in commercial broilers.

Effect of adjuvants on in ovo vaccination against Newcastle disease on hatchability, performance and antibody titres in commercial pullets.

This study was conducted to investigate the efficacy of in ovo administration of aluminium hydroxide (AH) and/or mannan oligosaccharide (MOS) adjuvants along with lentogenic VG/GA strain-Avinew to alleviate the embryonic pathogenicity of Newcastle disease virus. Six hundred and thirty fertilized Bovans eggs were divided into nine groups of 70 each incubated in a commercial hatchery and administered with eight types of in ovo injections in a factorial design of 2 × 2 × 2 including with/without AH, MOS and Newcastle disease vaccine (NDV), and one uninjected group on day 18 of incubation. Hatchability was higher in the eggs received MOS and/or AH adjuvants plus NDV compared those injected with NDV alone which confirmed the attenuation of NDV. However, the average daily feed intake and feed conversion ratio of pullets hatched from NDV-injected eggs were significantly reduced, but did not affect growth performance during 0-42 days of age. The performance of pullets hatched from eggs injected with AH, MOS or their mixture with NDV was not significantly different during all growth periods. Pullets from MOS + vaccine injected eggs had significantly higher antibody titres against NDV compared to those hatched from either injected with saline or uninjected on d 28 (p < .05). In addition, AH plus vaccine and MOS significantly improved total anti-SRBC and IgG respectively. Histological observation revealed that injection of MOS adjuvant into eggs led to increase crypt depth, whereas AH injection caused a reduction in villus surface area of jejunum in chicks on d 14 post-hatch. It is concluded that in ovo MOS injection as compared to AH may be more effective to attenuate the embryonic pathogenicity of in ovo NDV injection.

A bioinformatic approach to check the spatial epitope structure of an immunogenic protein coded by DNA vaccine plasmids.

In this study, we used an approach to check the Hemagglutinin antigen-antibodies interactions after fusion of one or two gene segments to Hemagglutinin gene in some influenza DNA vaccines. We designed different DNA vaccine constructs containing Hemagglutinin 9 (H9) gene fused to four or eight 29 amino acids of C3d (4/8P29C3d) and/or 3, 4 domains of the Fc part of IgY (FcIgY) coding sequences. As there are receptors for P29C3d and FcIgY on the immune cells, fused H9 are targeted to these cells. Three dimensional (3D) structures of the DNA vaccine coded proteins were modeled and docked with two antibodies (1KEN, 1QFU) to evaluate the effect of the H9 gene fusion to the other gene segments (4, 8 P29C3d and FcIgY) on the interaction of two H9 spatial epitopes. Also, we docked DNA vaccine proteins containing Fc IgY to its receptor (CHIR AB1) and compare interaction affinity of Fc IgY alone with affinity of DNA vaccines containing Fc IgY. The average of 1KEN and 1QFU interface scores were 94.89 and 93.09% of H9 DNA vaccine-antibodies interface scores, respectively. These percentages showed a little change in the H9 immunogenic parts. Also, because of spatial freedom of H9 part in all DNA vaccine proteins, added parts may not interfere with antibody-antigen interactions. Once, H9+FcIgY and CHIR AB1 affinity decreased in comparison with affinity of Fc IgY alone and CHIR AB1, affinity of H9+8P29C3d+FcIgY and CHIR AB1 increased to 132%. So, this would be expectable that despite of loss of affinity in H9 and its antibodies in the H9+8P29C3d+FcIgY, dramatic increase of Fc IgY and CHIR AB1 affinity in this group, could repair the loss of H9 affinity and may lead to a better immunogenicity.

Impact of milk components on recovery of viral RNA from MS2 bacteriophage.

Noroviruses are responsible for approximately 44 % of outbreaks involving dairy products for which causative agents are reported. Recovery of viruses from milk and dairy products is a difficult task. The role of different components of milk in the recovery of viral RNA was evaluated in this study. Four model milk formulations (A-D) were prepared by mixing different combinations of lactose, whey protein, casein, and fat in water. Each model formulation was spiked with five concentrations of bacteriophage MS2. The phenol-guanidine thiocyanate-chloroform protocol was used for extracting viral RNA from the model milk formulations and then extracted RNA was measured by a nanodrop spectrophotometer in ng/µl. The results showed that casein and whey protein had the highest negative impact on RNA yield, especially when the number of MS2 was less than 1.3 pfu/ml. The highest RNA recovery was obtained from the model milk formulation containing all four components; lactose, whey protein, casein, and fat. The amount of extracted RNA was closely correlated with the dry matter content of each formulation and the spiked concentration of coliphage using response surface modeling (R²:0.93). It was determined that milk fat is the most effective component in facilitating RNA extraction and the highest RNA yield can be achieved via elimination of whey protein and casein from milk by centrifugation at 40,000×g for 60 min. To achieve the highest viral RNA recovery efficiency by the proposed method, milk fat must be recombined with the supernatant of the centrifuged sample and then homogenized before performing the extraction protocol.

Study on ovine abortion associated with Toxoplasma gondii in affected herds of Khorasan Razavi Province, Iran based on PCR detection of fetal brains and maternal serology.

Toxoplasma gondii, an obligatory intracellular protozoan parasite, is one of the causative agents of ovine abortion, as reported in many countries. Different techniques are being used to detect this pathogen in infected ovine fetuses. One of the most sensitive and specific diagnostic techniques is Nested-PCR amplification of the B1 target gene of the organism. In total, 200 brain samples of aborted ovine fetuses and maternal sera submitted from different parts of Khorasan Razavi province, Iran were investigated to track the role of Toxoplasma gondii in ovine abortion by a slightly modified Nested-PCR and IFAT assays, respectively. Among all samples, 27 (13·5%) were PCR-positive and 31 (15·5%) were IFAT-positive and the Toxoplasma-induced abortion prevalence calculated was 8·8% to 18·2% with 95% confidence interval. Results show that high levels of congenital transmission may occur in 27/31(87%) of pregnancies with an excellent logical agreement (ĸ=0·9) between 2 different tests. According to the results of this study, the Nested-PCR employed in this investigation could be recommended as an applied routine test for the routine examination and confirmation of Toxoplasma gondii-induced ovine abortion.

Impact of milk components in recovery of the MS2 bacteriophage as an indicator of enteric viruses.

The objective of this study was to characterize the role of milk components in the recovery of viral particles from raw milk. For such characterization, four model milk formulations (A-D) were constituted by mixing different combinations of lactose, whey protein, casein, and fat into water. Each model formulation was spiked with six concentrations of bacteriophage MS2. The soluble and insoluble components of each model milk formulation were separated by centrifugation at 40,000 x g and viruses were enumerated in each supernatant fluid and pellet by the double agar layer (DAL) method. When samples were spiked with MS2 at concentrations lower than 4.8 x 10(5) pfu/ml, milk components did not significantly impact the overall recovery. However, the impact of milk components was measurable at higher concentrations. In general, higher numbers of MS2 were recovered from supernatant fluids of model milk formulations containing no fat. The highest number of viral particles were recovered from the pellet of model C (lactose+whey protein+casein). The recovery efficiency of MS2 was correlated with the dry matter contents of each model milk formulation and the initial spiking concentration of coliphage using response surface modeling.

Genetic diversity of beak and feather disease virus detected in psittacine species in Australia.

The complete nucleotide (nt) sequence of eight isolates of beak and feather disease virus (BFDV) obtained from a range of psittacine species with psittacine beak and feather disease (PBFD) from throughout Australia were compared with the sequences of two BFDV isolates previously reported from Australia (BFDV-AUS) and America (BFDV-USA), respectively. All isolates had the same basic structure including the position of the open reading frames, the hairpin structure between ORF1 and ORF2, the nonanucleotide motif (TAGTATTAC) therein, the three motifs of Rep protein encoded from ORF1 and involved in rolling circle replication, and the P-loop motif previously described, but the genome size of the eight isolates ranged from 1992 to 2018 nt. Overall nt identity of the isolates compared to BFDV-AUS ranged from 84 to 97%; the variation was due to a combination of point mutations and a number of deletions and insertions ranging from 1 to 17 nt in size detected in both coding and noncoding regions. The identity of the nt sequence of ORF2 compared to BFDV-AUS varied from 80 to 99%, while the identity of the deduced amino acid sequences varied from 73 to 99%. Phylogenetic analysis grouped the isolates into four clusters but there were no apparent regional differences or differences related to the psittacine species of origin. While seven ORFs with the potential to encode proteins greater than 8.7 kDa were detected in the BFDV-AUS isolate described previously, only three of these ORFs were detected in all 10 BFDV isolates for which sequence data were available. The three ORFs were ORF1 that presumably encodes the Rep protein, ORF2 presumably the major capsid protein, and the ORF previously designated ORF5. The ORF5 was of two size classes in different isolates, 303 and 474 nt, and only the first 303 nt of the viruses with an ORF of 474 nt were common to the other isolates.

A universal polymerase chain reaction for the detection of psittacine beak and feather disease virus.

A universal PCR assay was designed that consistently detected psittacine beak and feather disease virus (BFDV) in psittacine birds affected with psittacine beak and feather disease (PBFD) from different geographic regions across Australia. Primers within open reading frame 1 (ORF1) of the BFDV genome consistently amplified a 717 bp product from blood and/or feathers of 32 birds with PBFD lesions. The PCR did not amplify a product from the feathers or blood from 7 clinically normal psittacine birds. Primers based on regions outside of ORF1 did not consistently produce a PCR product, suggesting there was some genomic variation outside ORF1. The amplified ORF1 PCR products of 10 BFDV isolates, from different psittacine species and from various regions around Australia, were cloned and comparative DNA sequence analysis demonstrated 88-99% of the ORF1 fragments. The derived amino acid sequences of the amplified ORF1 fragments demonstrated similar identity between all 10 isolates. Within ORF1, there was complete conservation of the putative nucleotide binding site and marked conservation of 2 other motifs previously identified as essential components of the replication-associated proteins of other circoviruses and geminiviruses.

Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circoviruses, and chicken anaemia virus.

Cloning and sequencing of the circular, single-stranded DNA of one isolate of psittacine beak and feather disease virus (BFDV) demonstrate a genome composed of a circular molecule of 1993 nucleotide bases. An analysis of the assembled replicative form demonstrated seven open reading frames (ORFs) (three in the virion strand and four in the complementary strand), potentially encoding seven viral proteins of >8.7 kDa. High amino acid sequence similarity was demonstrated between a potential 33.3-kDa protein product of ORF1 of BFDV and the replicase-associated protein of porcine circovirus (PCV), subterranean clover stunt virus, and faba bean necrotic yellows virus. However, significant similarity in nucleotide or amino acid sequences was not present between BFDV and chicken anaemia virus. A potential stem-loop structure similar to that found in PCV and plant circoviruses was present in the putative encapsidated strand of the BFDV genome. At the top of this structure, a nonanucleotide motif (TAGTATTAC) similar to that of PCV, plant circoviruses, and geminiviruses also was recognised. Comparison of the deduced amino acid sequences of ORF2 of BFDV and PCV demonstrated 29.1% identity, and in both viruses, ORF2 is located on the complementary strand, beginning close to or within the hairpin stem. Our findings provide further evidence of a close relationship among BFDV, PCV, and plant circoviruses but not chicken anaemia virus.