The arrival of highly pathogenic avian influenza viruses H5N8 in Iran through two windows, 2016.

The highly pathogenic avian influenza (HPAI) H5N1 virus has received considerable attention during the past 2 decades due to its zoonotic and mutative features. This Virus is of special importance due to to the possibility of causing infection in human populations. According to it's geographical location, Iran hosts a large number of aquatic migratory birds every year, and since these birds can be considered as the host of the H5 HPAI, the country is significantly at risk of this virus. the In this study, the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) genes of the H5N8 strain were identified in Malard county of Tehran province and Meighan wetland of Arak city, Markazi province were investigated. Based on the analysis of the amino acid sequence of the HA genes, the cleavage site of the gene includes the PLREKRRKR/GLF polybasic amino acid motif, which is a characteristic of highly pathogenic influenza viruses. The HA gene of two viruses had T156A, S123P, S133A mutations associated with the increased mammalian sialic acid-binding, and the NA gene of two viruses had H253Y mutations associated with the resistance to antiviral drugs. Phylogenetic analysis of the HA genes indicated the classification of these viruses in the 2.3.4.4 b subclade. Although the A/Goose/Iran/180/2016 virus was also an H5N8 2.3.4.4 b virus, its cluster was separated from the A/Chicken/Iran/162/2016 virus. This means that the entry of these viruses in to the country happened through more than one window. Furthermore, it seems that the introduction of these H5N8 HPAI strains in Iran probably occurred through the West Asia-East African flyway by wild migratory aquatic birds.

Identification of Cytotoxic T lymphocyte (CTL) Epitope and design of an immunogenic multi-epitope of Bovine Ephemeral Fever Virus (BEFV) Glycoprotein G for Vaccine Development.

Bovine ephemeral fever (BEF), a vector-borne disease of cattle and water buffalo, is enzootic in tropical and subtropical zones of Asia, Australia, and Africa. Since cytotoxic T lymphocytes (CTL) responses may play a key role in the control of bovine ephemeral fever virus (BEFV) infection, it is important to identify and characterize the CTL target epitopes of BEFV antigens. The current study has been designed to identify and characterize the potential CTL epitopes using the Immuno-informatics tools, and it helped find the potent vaccine candidates against BEF. Antigenicity, toxicity, allergenicity, and immunogenicity testing of predicted CTL epitopes was done. Total four CTL epitopes for BEFV G protein, have been identified as potential epitopes. Prediction of the 3D structure of multi-epitope (final structure) was performed using I-TASSER server. Model 1 was selected as the best model with C-Score: -3.71. The modeled G protein structure and multi-epitope structure were validated by the Ramachandran plots Prosa and Verify 3D server. Epitopic regions of 3D protein structure were identified by Chimera UCSF software. Physicochemical properties of the Multi epitope were evaluated using ProtParam server. This is the first report of CTL epitope in the G protein of BEFV. In this manner, they would play an important role in evoking the immune response as well as vaccine development. However, in vitro and in vivo experimental studies are required for suggested epitopes verification. The multi-epitope was designed from regions of the G protein sequence that lacked mutation and genomic diversity. Therefore, it can be introduced as a protein vaccine from all strains of BEFV as a vaccine candidate for design.

Pasteurella multocida PlpE Protein Polytope as a Potential Subunit Vaccine Candidate.

Pasteurella multocida is the causative agent of a range of animal, and occasionally human, diseases. Problems with antimicrobial treatment of P. multocida highlight the need to find other possible ways, such as prophylaxis, to manage infections. Current vaccines against P. multocida include inactivated bacteria, live attenuated and nonpathogenic bacteria; they have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence. Using bioinformatics approaches, potentially immunogenic and protective epitopes were identified and merged to design the most optimally immunogenic triple epitope PlpE fusion protein of P. multocida as a vaccine candidate. This triple epitope (PlpE1 + 2 + 3) was cloned into the pBAD/gIII A plasmid (pBR322-derived expression vectors designed for regulated, secreted recombinant protein expression and purification in Escherichia coli), expressed in Top 10 E. coli and purified in denatured form using Ni-NTA chromatography and 8 M urea. The immunogenicity of the purified proteins in BALB/c mice was assayed by measuring immunoglobulin G (IgG) responses. The protection potential was evaluated by challenging with 10 LD50 of serotype A:1, X-73 strain of P. multocida and compared with commercially available inactivated fowl cholera vaccine and PlpE protein. IgG levels elicited by the polytope fusion protein of P. multocida PlpE were higher than both commercially available inactivated fowl cholera vaccine and PlpE protein. Surprisingly, protection was independent of IgG level; commercially available inactivated fowl cholera vaccine (100% protection) was more protective than the polytope fusion protein (69% protection) and PlpE protein (69% protection). These results also confirm that IgG level is not a reliable indicator of protection. Further studies to evaluate the other antibody classes, such as immunoglobulin A or M, are required. The role of cell-mediated immunity should also be considered as a potential protection pathway.

Effects of dietary turmeric (Curcuma longa) on innate and acquired immune responses in broiler chicken.

One of the most desired treatments to combat stress and inflammation with minimal adverse effects in large bird populations is food additives. This study investigated the effects of dietary turmeric (Curcuma longa) on the level of serum amyloid A (SAA) as an indicator for acute phase response and antibody titer to Newcastle vaccine as an indicator for humoral immune response. A total of 300 Ross broiler chickens were assigned to five dietary groups. Two treatments received basal diets supplemented with different amount of turmeric (250 and 500 mg/kg). One group received aspirin (ASA; 250 mg/L) and one group aspirin-vitamin C (ASA; 250 mg/L + Ascorbic acid; 20 mg/L) in drinking water. There was one control group that received no feeding additives. The levels of SAA and humoral antibody response to Newcastle vaccine were measured during the entire production period. Turmeric administration significantly decreased the serum SAA concentrations after 2 weeks of treatment and later. It also significantly reduced SAA elevation due to the vaccinations on day 17 but not on day 28. After the second vaccination (d 19) ELISA titer in all treatment groups was higher than control group. Significant effect of dietary turmeric on body weight was also found at week 3 and later ages. Administration of 250 mg turmeric per kg diet is recommended for broiler chickens. It is concluded that turmeric is beneficial to minimize inflammatory effects of vaccination in commercial broiler chickens. Turmeric prevents and reduces stress and negative effects of inflammation and stimulates growth performance of broiler chickens.

In silico Analysis of Pasteurella multocida PlpE Protein Epitopes As Novel Subunit Vaccine Candidates.

Background: Pasteurella multocida is a Gram-negative, non-motile, non-spore forming, and aerobic/anaerobic cocobacillus known as the causative agent of human and animal diseases. Humans can often be affected by cat scratch or bite, which may lead to soft tissue infections and in rare cases to bacteremia and septicemia. Commercial vaccines against this agent include inactivated, live attenuated, and non-pathogenic bacteria. Current vaccines have certain disadvantages such as reactogenicity or reversion to virulence. Therefore, the aim of this study was to reach a multi-epitope vaccine candidate that could be serotype independent and covers most incident serotypes of P. multocida. Methods: In this study, reverse vaccinology strategy was used to identify potentially immunogenic and protective epitopes. First, multiple alignments of different sequences of Pasteurella lipoprotein E (PlpE) from various serotypes of P. multocida were analyzed to identify the conserved regions. Bioinformatics tools were then applied to predict and select epitopes for further studies. Results: Three different conserved immunogenic regions were selected according to the selected criteria, and their various sequential orders were evaluated structurally by in silico tools to find the best order. Conclusion: In searching the epitopes of PlpE to design a new vaccine candidate against pasteurellosis, we found the region 1 + region 2 + region 3 (without any linker between regions) of epitope, including the regions of PlpE protein of P. multocida, as the appropriate serotype independent vaccine candidate against pasteurellosis.

Pasteurella multocida Vaccine Candidates: A Systematic Review.

Pasteurella multocida (P. multocida) is the highly contagious causative agent of a broad range of diseases in animals as well as an occasional human pathogen. Economically significant infections caused by P. multocida include avian fowl cholera, rabbit snuffles, and hemorrhagic septicemia in cattle, goats and pigs. Chemotherapy of pasteurellosis infections has some limitations, such as high cost of treatment, low efficacy, and the possibility of therapy failure due to antibiotic resistance. Prophylactic immunization offers a safe and effective preventive measure in case of zoonotic diseases. Bacterins, live attenuated and some old traditional vaccines against pasteurellosis remain in use today, beside their limitations. However, the past few years have seen significant progress in research to identify modern, effective vaccine candidates, but there is no new vaccine produced by new strategies. While scientists should struggle with a lot of aspects to design vaccine producing strategies, this review shows how pasteurellosis vaccine evolved and the limitations in its application which need to be overcome.

Comparison of cattle BoLA-DRB3 typing by PCR-RFLP, direct sequencing, and high-resolution DNA melting curve analysis.

Major histocompatibility complex (MHC) represents an important genetic marker for manipulation to improve the health and productivity of cattle. It is closely associated with numerous disease susceptibilities and immune responses. Bovine MHC, also called bovine leukocyte antigen (BoLA), is considered as a suitable marker for genetic diversity studies. In cattle, most of the polymorphisms are located in exon 2 of BoLA-DRB3, which encodes the peptide-binding cleft. In this study, the polymorphism of the BoLA-DRB3.2 gene in Holstein's calves was studied using high resolution melting curve analysis (HRM). Observed HRM results were compared to PCR-RFLP and direct sequencing techniques. Eight different HRM and seven different RFLP profiles were identified among the population studied. By comparing to sequencing data, HRM could completely discriminate all genotypes (eight profiles), while the RFLP failed to distinguish between the genotypes *1101/*1001 and *1104/*1501. According to the results, the HRM analysis method gave more accurate results than RFLP by differentiating between the BoLA-DRB3.2 genotypes. Due to the Co-dominant nature of the MHC alleles, HRM technique could be used for investigating the polymorphisms of genotypes and their associations with immune responses.

Molecular characterization of infectious bronchitis virus based on RNA-dependent RNA polymerase gene.

Extensive rate of variations in the S1 gene (spike glycoprotein subunit gene) of infectious bronchitis virus (IBV) causes challenges for clinicians in counting variants for differentiation of infected from vaccinated birds and addressing the variants of unknown significance. This study investigated the possibility of using an RNA-dependent RNA polymerase gene (RdRp) as a target for molecular characterization of IBV strains in Iran. Trachea samples were collected from commercial broiler flocks (n = 52) showing respiratory syndrome. Specific PCR primers were designed for a variable region located in the RdRp gene flanked by highly conserved regions. Reverse transcriptase PCR followed by sequence analysis identified eight IBV variants, with an overall prevalence of 44.2%. Deduced nucleotide and amino acid sequences were compared with published sequences for IBV strains. Because of the long-distance similarities, the field samples could be discriminated from vaccine strains. Phylogenetic analysis of RdRp gene sequences resulted in clustering of the IBV strains related to each area. Using RdRp as a genetic marker eliminates the challenges arising from the enormous variations that make it difficult to discriminate between field and vaccine strains as well as affiliate certain variants to various geographical areas.

Correction: Effects of vaccination on acute-phase protein response in broiler chicken.

[This corrects the article DOI: 10.1371/journal.pone.0229009.].

Effects of vaccination on acute-phase protein response in broiler chicken.

Broiler chickens experience an acute-phase response (APR) through vaccination, which reflects the innate immunity and stress related to immunization. It is also considered that APR can modulate adaptive immunity and response to infection. As biomarkers for APR, assessing the acute-phase proteins (APPs) function and their levels in response to immunization is of great value for vaccine design, development and administration. In this study, the heterophils/lymphocyte (H/L) ratio and the level of APPs was evaluated in broilers with three different Newcastle disease (ND) vaccination regimens. Inactivated ND vaccine (IND) was administered by the intramuscular route. Live attenuated strains, Lasota and Vitapest, was administered by ocular routes. H/L ratio, serum amyloid A (SAA) and alpha-1 acid glycoprotein (AGP) were measured before and after two rounds of vaccination on days 10 and 21. In a comparison between the three vaccines, H/L ratio in IND group significantly increased to 3 fold (1.48 ± 0.41) after the first vaccination while the Lasota and Vitapest showed a milder response. The concentration of SAA increased after 24h by 1.8-fold in IND group (0.116 ± 0.015 mg/L) and 2-fold in Lasota group (0.14 ± 0.002 mg/L). Significant changes were found in Vitapest group after 48h post vaccination (0.113 ± 0.016 mg/L). Elevation pattern of AGP, 24 hours after first vaccination in IND (3.5-fold) and Vitapest (2.5-fold) was different from Lasota in which the peak was reached after 48 hours (2.9-fold). Except for IND group, no significant changes in SAA and AGP concentrations were detected after the second vaccination. A significant positive correlation between SAA values at day 22 and HI titers at day 28 (r = 0.998, P≤0. 0.005) was found. According to these results, different types of ND vaccines can cause different patterns of acute phase responses. Assessment of stress and level of acute-phase proteins can be used for prediction of immune response outcomes in vaccine design and development.

In silico prediction of linear B-cell epitopes for S1 protein of two Iranian 793/B isolates and their changes after 90 serial passaging.

Neutralizing, serotype-specific, and hemagglutination-inhibiting antibodies against infectious bronchitis virus (IBV) are induced by epitopes in the S1 protein. Most changes in the virus genome due to mutation and recombination during serial passaging in embryonated chicken eggs occur in the S1 gene. In the current study, we tried to predict the potential linear B-cell epitopes of the S1 subunit of two Iranian 793/B isolates and then we analyzed their changes at passage level 90 due to mutations at this passage level. To predict linear B-cell epitopes of the S1 protein belonging to two Iranian 793/B isolates, we used two online epitope prediction programs called BepiPred and ABCpred. Some of the most important features of proteins including antigenicity, physicochemical properties, and secondary structure composition were analyzed. The predicted epitopes were studied between wild viruses and their passage level 90 viruses. We identified 15 potential linear B-cell epitopes among which six epitopes had the highest scores of physicochemical properties and antigenicity. Due to amino acid substitutions, seven predicted epitopes had different amino acid sequences at passage level 90. Among eight epitopes with no amino acid substitution at passage level 90, three epitopes had the highest scores. These three conserved epitopes including NH2-NQLGSCPLTGMI-COOH, NH2-GNFSDGFYPFTNSSLVKD-COOH, and NH2-GPIQGGC-COOH might be strategic and potential candidates for use in designing epitope-based vaccine researches. In conclusion, based on scores of physicochemical properties and antigenicity, it seemed that the sequence of most epitopes in wild viruses might be more antigenic and immunogenic compared to their sequence in viruses of passage 90.

Evaluation of humoral immune responses to enterotropic lentogenic VG/GA vaccine of Newcastle disease in commercial turkey poults (Meleagris gallopavo).

BACKGROUND: Villegas-Glisson/University of Georgia (VG/GA) strain of Newcastle disease virus (NDV) is recommended for the initial vaccination of commercially reared turkey poults. However, the vaccine-induced antibody responses have not been studied in this species. The level of systemic humoral immune responses against the NDV was investigated in commercial turkey poults vaccinated with the VG/GA vaccine. One hundred eighty-two hybrid strain of turkey poults (Meleagris gallopavo) were divided randomly into vaccinated and unvaccinated groups. The vaccinated group was given the VG/GA vaccine at 10 and 20 days of age. To investigate the vaccine immunity, the level of specific IgY and IgA in serum samples were determined using ELISA and haemagglutination inhibition assays (HI). The biological half-life of maternal antibodies was also determined before the immunization. RESULTS: VG/GA-specific antibodies were detected in the vaccinated turkey poults and were significantly higher in the vaccinated group compared to the unvaccinated group. IgY and IgA antibodies showed a significant increase in titers 14 days after the second vaccination and reached a peak on day 35 of age. The correlation coefficient and intra-rater reliability showed a significant correlation between the HI titers and IgY/IgA ELISA values. Maternal IgY and IgA levels were found to decline in the serum with half-lifes of 7.68 ± 2.35 and 2.18 ± 0.82 days, respectively. CONCLUSIONS: Enterotropic lentogenic VG/GA vaccine induced a marked humoral immune response against the NDV in turkey poults. The positive correlation between IgY and IgA highlights the role of these two antibody classes in controlling the Newcastle disease in turkey poults.

Molecular characterization of enterotoxigenic Escherichia coli isolated from neonatal calves diarrhea.

Enterotoxigenic Escherichia coli (ETEC) produces different virulence factors allowing the bacterium to colonize and develop watery diarrhea. Proteomics studies have also introduced new protein belonging to the secretion pathways, antigen 43 (Ag43), which plays important role in E. coli pathogenesis. The objective of this study was to investigate O-types and virulence factors of E. coli isolates from neonatal calves diarrhea. Total of 120 isolates from diarrheic calves were genotyped for their O groups and the presence of virulence genes K99, F41 and STa as well as Ag43. The predominant O-type was O101 (51.00%) and the prevalence of K99, F41 and STa was 7 (5.80%). The Ag43 was detected in all samples with three different allelic patterns. Our results indicated that K99 positive isolates certainly have one of each 2200 bp or 1800 bp or both copies of Ag43 passenger domain, while negative K99 isolates lack the Ag43. The results reported here provide informative data regarding the prevalence of E. coli O-types and their virulence factors in enteric colibacillosis. The Ag43 that was more found in K99 positive isolates might be associated with diarrhea-causing E. coli strains in neonatal calves.

Evaluation of a new lymphocyte proliferation assay based on cyclic voltammetry; an alternative method.

Lymphocyte proliferation assays are widely used to assess the cell-mediated immunity. Current in vitro testing methods that are being used have extensive applications but still more problematic, due to the technical complexity and the needs for specialized equipment and reagents. Electrochemical methods such as cyclic voltammetry represent a very promising tool for the development of label-free in vitro assays of cell proliferation and viability. Here, a novel procedure based on voltammetric behaviours of proliferating cells was fabricated. Results indicated that proliferation in cell cultures and whole blood can be monitored electrochemically using cyclic voltammetry. In the comparison with colorimetric (MTT) assay, cyclic voltammetry gave the best correlation with cell count data over a range of 1200-300,000 cells/well of a microplate. Besides the advantages of short assay duration (4 hours) and the rapidness, the possibility use of fresh blood without further processing, would give more accurate results because cells are monitoring in an intact environment. Cyclic voltammetry assay is an efficient analytical method, which can provide a simple platform for the electrochemical study of lymphocyte proliferation.

Association of MHC class II haplotypes with reduced faecal nematode egg count and IgA activity in British Texel sheep.

Nematode infection is one of the principal diseases suffered by sheep and the class II region of the MHC has been repeatedly associated with differences in susceptibility and resistance to infection. The aim of this study was to examine the association of MHC class II haplotypes in a flock of Texel sheep with faecal egg counts and antibody responsiveness. Two haplotypes carried the DRB1*11:01 allele which has previously been associated with reduced egg counts in Scottish Blackface and Suffolk sheep. One of the two haplotypes was associated with reduced egg counts in the Texel breed, and both haplotypes were associated with reduced IgA activity against an extract from fourth-stage larvae. The reduced IgA activity is probably a consequence of reduced numbers of fourth-stage larvae in sheep carrying the resistance allele. The association of specific MHC alleles with reduced egg counts, reduced worm numbers and decreased IgA activity provides a mechanism for the density-dependent regulation of parasite growth and fecundity.

Protection against neonatal enteric colibacillosis employing E. Coli-derived outer membrane vesicles in formulation and without vitamin D3.

OBJECTIVE: Enterotoxigenic Escherichia Coli (ETEC) is the cause of diarrhea and even death in humans and offspring of animals. Outer membrane vesicles (OMVs) of the ETEC was prepared and its potential as a vaccine candidate against enteric colibacillosis in neonatal mice was evaluated. Dam mice intradermally injected with ETEC-derived OMVs and OMVs plus an active form of vitamin D3 (avD3). Mucosal and systemic immune responses in mice and passive immunity protection against ETEC lethality in their offspring was investigated. RESULTS: Immunization of adult mice via ETEC-derived OMV alone and in formulation with avD3 protect offspring from ETEC-induced lethality. Nevertheless, avD3 did not indicate a positive effect on mucosal and systemic immune responses. Only the combination of OMV plus avD3 elicited a significant (P < 0.05) increase in the level of specific IgA antibodies in serum.

LEI0258 microsatellite variability and its association with humoral and cell mediated immune responses in broiler chickens.

Major histocompatibility complex (MHC) has a profound influence on disease resistance or susceptibility, productivity and important economic traits in chicken. Association of the MHC with a wide range of immune responses makes it a valuable predictive factor for the disease pathogenesis and outcome. The tandem repeat LEI0258 is a genetic marker which is located within the B locus of chicken MHC and strongly associated with serologically defined haplotypes. LEI0258 microsatellite marker was applied to investigate the MHC polymorphism in Ross 308 broiler chicken (N=104). Association of LEI0258 alleles with humoral and cell mediated immune responses to Newcastle disease (ND), Infectious bursal disease (IBD) and Avian influenza (AI) vaccines were also examined. LEI0258 polymorphism was determined by PCR-based fragment analysis, and association of LEI0258 alleles with immune responses were evaluated using multivariate regression analysis and GLM procedures. A total of seven alleles ranging from 195 to 448bp were found, including two novel alleles (263 and 362bp) that were unique in Ross 308 broiler population. Association study revealed a significant influence of MHC alleles on humoral and cellular immune responses in Ross population (P<0.05). Alleles 385 and 448bp were associated with increased peripheral blood lymphocyte proliferation response. Alleles 300, 362 and 448bp had a positive effect on immune responses to Infectious bursal disease vaccine, and allele 263bp was significantly correlated with elevated antibody titer against Newcastle disease vaccine. Results obtained from this study confirmed the important role of MHC as a candidate gene marker for immune responses that could be used in genetic improvement of disease-resistant traits and resource conservation in broiler population.

Association of BoLA-DRB3.2 Alleles with BLV Infection Profiles (Persistent Lymphocytosis/Lymphosarcoma) and Lymphocyte Subsets in Iranian Holstein Cattle.

Major histocompatibility complex (MHC) is the best-characterized genetic region associated with resistance and susceptibility to a wide range of diseases. In cattle, the most important example of the relationship between the MHC and infectious diseases has been established by the resistance to Bovine leukemia virus (BLV) infection. The association of the bovine MHC class II BoLA-DRB3.2 alleles with BLV infection profiles was examined. BoLA-DRB3.2 allelic diversity was determined in 190 Iranian Holstein cattle using direct sequencing method. Association of the DRB3.2 alleles with BLV infection profiles was found as the odds ratio. Effects of the alleles on lymphocyte subsets were also evaluated by multivariate regression analysis and GLM procedures. The studied cattle were categorized into three groups: BLV seronegative, BLV seropositive with persistent lymphocytosis (PL), and BLV seropositive with lymphosarcoma (LS). The PL profile was significantly associated with the BoLA-DRB3.2*0101, *1101 and *4201 alleles, although the *3202 allele mediating resistance to PL was observed. Significant association was found between the BoLA-DRB3.2*1802, *3202, and *0901 alleles and susceptibility to LS, while the *0101 and *1101 alleles were associated with resistance to LS. BoLA-DRB3.2 alleles also showed a significant correlation with CD4, CD8, CD21 cells and CD4/CD8 ratio. Allelic differences influence the immune response to BLV infection and developing the disease profile. These differences also have important consequences for tumor resistance.

Subcutaneous immunization with a novel immunogenic candidate (urease) confers protection against Brucella abortus and Brucella melitensis infections.

Brucellosis is a world prevalent endemic illness that is transmitted from domestic animals to humans. Brucella spp. exploits urease for survival in the harsh conditions of stomach during the gastrointestinal infection. In this study, we examined the immune response and the protection elicited by using recombinant Brucella urease (rUrease) vaccination in BALB/c mice. The urease gene was cloned in pET28a and the resulting recombinant protein was employed as subunit vaccine. Recombinant protein was administered subcutaneously and intraperitoneally. Dosage reduction was observed with subcutaneous (SC) vaccination when compared with intraperitoneal (IP) vaccination. rUrease induced mixed Th1-Th2 immune responses with high titers of specific IgG1 and IgG2a. In lymphocyte proliferation assay, splenocytes from IP and SC-vaccinated mice displayed a strong recall proliferative response with high amounts of IL-4, IL-12 and IFN-γ production. Vaccinated mice were challenged with virulent Brucella melitensis, B. abortus and B. suis. The SC vaccination route exhibited a higher degree of protection than IP vaccination (p value ≤ 0.05). Altogether, our results indicated that rUrease could be a useful antigen candidate for the development of subunit vaccines against brucellosis.