Efficacy of Echinacea purpurea and protexin on systemic and mucosal immune response to Newcastle diseases virus vaccination [VG/GA strain] in commercial turkey poults

Background: it is important to understand the efficacy of immunoregulatory materials, herbal remedies or probiotics, in different parts of immune system following vaccination with different tropism

Objectives: aim of this study was to evaluate the effect of Echinacea purpurea and a probiotic [protexin] on systemic and mucosal immune response in turkey

Methods: a total of 288 1-day-old male turkey poults were randomized into 6 groups as follow: Group T1: Turkeys received Echinacea purpurea at the rate of 1 ml /1 liter water and Newcastle disease virus [NDV] vaccine, Group T2: Turkeys received probiotic at the rate of 1 g /1 liter water and NDV vaccine, Group T3: Positive control, turkey received NDV vaccine without any additives. Group T4: Turkeys received Echinacea purpurea at the rate of 1 ml /1 liter water without NDV vaccine. Group T5: Turkeys received probiotic at the rate of 1 g /1 liter water without NDV vaccine, Group T6: Negative control group, neither vaccinated against NDV vaccine nor given additives. At age of 10 and 20 days, poults were vaccinated with Villegas-Glisson/University of Georgia [VG/GA] strain of Newcastle disease vaccine by eye dropper method. For systemic and mucosal antibody analyses, blood samples and tracheal lavages were collected at different ages. The titers of antibody against NDV were measured using ELISA and HI tests

Results: addition of Echinacea to the water increased the systemic IgG, IgA and HI compared to the positive control group. Protexin supplementation to the water of T2 turkeys increased serum IgG and both total and specific IgA compared to the T3 group turkeys. Generally, turkeys that were supplemented with probiotic had higher specific and total tracheal IgA antibody levels than the other vaccinated groups. Among vaccinated turkeys only T1 group showed significantly higher HI antibody titers on day 42

Conclusions: results indicated that systemic and mucosal immunity of turkeys following vaccination against Newcastle disease [ND] could be improved by supplementation of Echinacea and probiotic. The effect of Echinacea purpurea on systemic immunity of turkeys seemed more pronounced than on mucosal immunity; further, the effect of probiotic on mucosal immunity was more obvious

[Allelic segregation of major histocompatibility complex using LEI0258 microsatellite marker in indigenous and commercial chickens]

Background: Major histocompatibility complex [MHC] in chicken has profound influence on resistance/susceptibility to disease, and production and reproduction traits. Microsatellite marker LEI0258 is a genetic indicator for MHC haplotypes. Recognizing diversity of MHC haplotypes in selectively bred populations will be helpful for selecting population resistant to disease and development of effective vaccines

Objectives: The purpose of the present study was to evaluate polymorphism at MHC in two populations of Khorasan indigenous chickens and commercial Leghorn breed using microsatellite marker LEI0258 and to investigate its segregation and heredity

Methods: A total of 335 blood samples from Khorasan Razavi indigenous chickens and commercial Leghorn population including parents [P] and offspring [F1], were analyzed. The MHC genotypes were determined using LEI0258 microsatellite. The study of allele heredity from P to F1 and Hardy-Weinberg equilibrium were conducted using Chi-square and Likelihood Ratio tests

Results: In Khorasan indigenous chickens 20 different alleles were identified for LEI0258 microsatellite. The allele 321 bp had the highest [22.88%] and the allele 182 bp had the lowest [0.16%] frequency. In the commercial population [Leghorn breed] 3 alleles were found for this marker of which the allele 261 bp had the highest [50%] and alleles 487 bp had the lowest [6%] frequency. In allele heredity analysis and Hardy-Weinberg equilibrium of Khorasan population, no significant differences were observed between P and F1 progenies

Conclusions: These results indicate a higher genetic variation in indigenous chickens compared to commercial breed. There was no preference for a particular allele in indigenous chickens. The higher frequency of some alleles in F1 population is due to the high frequency of the same alleles in parent population which their gametes make the population gene pool

[Study of the association of major histocompatibility complex with antibody response to vaccines in Khorasan native chickens]

Background: Major histocompatibility complex [MHC] plays a central role in regulation and control of the immune responses to infectious diseases. Due to its polymorphism, individual differences in response to vaccines have been observed in different chicken populations. Studying the association of chicken MHC with immune response to vaccines will help the control of infectious disease and vaccination success

Objectives: The present study aimed to evaluate the MHC polymorphism and its association with antibody response against infectious bursal disease [Gumboro], Newcastle [ND] and Influenza [AI] vaccines in Khorasan native chickens

Methods: Diversity of LEI0258 microsatellite marker [MHC genotyping] was investigated by fragment analysis method. Antibody titer against IBD was measured by ELISA and antibody titers against ND and AI vaccines were measured by Haemaglutination Inhibition [HI] assay. Statistical analysis was performed using SPSS software [version 21]. Univariate regression analysis was performed using weighted least squares with weight number of progeny mean data

Results: Total of 13 LEI0258 microsatellite alleles were identified in Khorasan native chickens which indicated a high genetic diversity in the population. The allele 361 bp had the highest [28.48%] and the allele 350 bp had the lowest [0.69%] frequency, respectively. In evaluating the association of MHC with immune responses, 311 and 313 bp alleles were significantly associated with elevated immune responses to Newcastle vaccine, while allele 266 bp was associated with lower IBDV antibody titers [p<0.05]

Conclusions: According to the important role of MHC in controlling infectious disease resistance or susceptibility and quality of immune responses, these results could be used for selection and improving the populations under selective breeding

Detection of gene expression and sequence analysis of chicken class II trans activator [CIITA]

Class II transactivator [CIITA] is a dominant transcriptional element, controlling numerous genes in the immune system. CIITA is expressed in a constitutive pattern in antigen presenting cells although its expression can occur in other cell types. Since the revelation of CIITA, there have been considerable advances toward understanding its role as an activator of MHC II genes in humans and mice; nonetheless, there is a lack of published data for this gene in other animals such as chickens. The goals of this study were to determine the expression of class II transactivator [CIITA] in chicken and analysis of the CIITA gene sequence between four Iranian indigenous chicken ecotypes. After securing the research accuracy and optimization of reaction conditions, cDNA and DNA samples of gene were obtained from four Iranian indigenous chicken ecotypes. The PCR and RT-PCR products were sequenced and the data were analyzed by bioinformatics software. Comparison of the sequencing results with the reference sequence of the red jungle fowl revealed that these sequences belonged to the predicted CIITA gene. There was a high conservation rate in the sequence of CIITA. Our results indicated that like other species, CIITA is transcripted in chickens' immune system cells. Further studies on chickens must be done to reveal CIITA roles in immune responses of chickens

[Developmental changes in Endothelin-1 and Endothelin type A receptor expression in the lungs of chickens [Broiler versus layer]]

BACKGROUND: Broilers lung mechanisms that regulate endothelin [ET] in the lung are complex and poorly understood

OBJECTIVES: METHODS: In this experiment lung ET-1 mRNA levels and lung mRNA expression for the ET[A] receptors were determined in lung tissue weekly [term = 42 days, intervals = 7 days] Serum endothelin concentration was also measured at these ages

RESULTS:The study showed that expression of endothelin in lungs of layers and broilers was similar during the first three weeks and the overall trend of ET-1 expression was increasing. However, there was a significant increase of ET-1 expression which started from the fourth week and gradually increased until the end of the commercial life of the chicken [day 42]. ET-A expression in the lungs of broilers was significantly higher than layers during the last three weeks of life. Overall, trends of serum ET-1 concentration increased in both layers and broilers, but serum ET-1 concentration in broilers was significantly higher than layers

CONCLUSIONS: The higher level of serum Endothelin and expression of ET-1 and ETAin broiler lungs may explain the higher sensitivity of broilers to the vasoconstrictions activity of endothelin and the higher sensitivity of these animals to pulmonary hypertension [PH]

[Association between amino acid sequences in peptide binding region [BOLA-DRB3] and susceptibility or resistance to calf diarrhea]

The major histocompatibility complex genes [mhc] encode MHC I and II molecules which present peptide fragments to T cells. Therefore these polymorphic molecules critically influence susceptibility to infectious diseases. At present study potential relationship between amino acid sequences in the antigen binding groove of different BoLA-DRB3 alleles and susceptibility or resistance to calf diarrhea was investigated. Twelve different DRB3 alleles were found among 171 calves [84 diarrheic and 87 healthy] analyesd by PCR-RFLP method. Amino acid sequences of the encoded peptide binding region were compared. 26 polymorphic positions were detected in this region. A significant association [p<0.05] was shown between occurrence of diarrhea and the presence of glutamic acid and tyrosine inpocket 4 and valine, glutamine and leucine in pocket 9 of peptide binding region. Thus it can be concluded that pockets 4 and 9 of the BoLA-DRB3 molecule would be involved in conferring susceptibility of calf to diarrhea

Exon 2 Ovar-DRB1 gene polymorphism in the Iranian Sangsari sheep

The major histocompatibility complex [MHC] plays a central role in the control of disease resistance and immune response. Extensive genetic diversity in MHC genes provides a valuable source for genetic improvement, via selection, in many domestic animals. Exon 2 of the class II MHC, termed Ovar-DRBl in domestic sheep [Ovis aries] has been suggested as important disease resistance and immune response gene. We characterized Ovar-DRBl in DNA samples from 138 individuals of a population of the Iranian Sangsari sheep breed using PCR-RFLP. Eight DRB1 alleles were identified among Iranian Sangsari sheep, including one previously unrecognized allele. Eight homozygous genotypes were observed: a, b, c, d, f, g, h andN. Genotype bb was the most common pattern [46 of 138]. Heterozygous genotypes [ag, cb, cd, bf, and bN] were also observed. The observed homozygosity and heterozygosity values were 0.6377 and 0.3623, respectively, vs expected values of 0.220 and 0.779. Iranian Sangsari population deviate significantly from the theoretical proportions [FIS = 0.5283; p = 0.0005]. In conclusion, PCR-RFLP analysis allows rapid identification of Ovar-DRBl types and discrimination of homozygous and heterozygous genotypes. This study indicates that the exon 2 region of the Ovar-DRBl gene is highly polymorphic in the Iranian Sangsari sheep breed

Serological and genomic detection of bovine leukemia virus in human and cattle samples

Bovine leukemia virus [BLV] is a retro virus responsible for lymphoproliferative disorders in cattle. Although infections of BLV in animals are well known, little is known about its capacity to infect humans. This study investigated the presence of anti-BLV antibodies and BLV pro viruses in human and cattle samples. An indirect enzyme-linked immunosorbent assay [ELISA] was used to detect anti-BLV antibodies while nested PCR was employed to identify BLV provirus sequences. The overall prevalence of anti-BLV antibodies in human and cattle samples were 12.50% and 16.73%, respectively. When using ELISA as a reference test, sensitivity and specificity for nested PCR were 0.625 and 0.970, respectively. The predictive value of a positive test was 0.862 and the predictive value of a negative test was 0.897. The percentage of cattle correctly classified by nested PCR assay was 89.1%. Nested PCR and Southern blot analysis, using primers specific for BLV gag sequences, revealed that BLV pro viruses were detectable in cattle and human samples. Our results highlight the risk of human exposure to BLV and the need for further investigations to determine whether BLV infection poses a health hazard for humans

Cloning and high level expression of bovine interferon gamma gene in eukaryotic cells [COS-7]

Interferon gamma [IFN-gamma] is one of the key cytokines in defining T helper 1 lymphocyte immune responses. In this study, the bovine IFN-gamma gene was cloned from spleen tissue RNA using the reverse transcription-polymerase chain reaction [RT-PCR]. IFN-gamma cDNA was sub-cloned and expressed in mammalian expression plasmid [pcDNA3.1[+]] under the control of the human cytomegalovirus [CMV] promoter. The predicted amino acid [aa] sequence of bovine IFN-gamma compared with corresponding known sequence from bovine [Bos taurus] was 100% identity and with ovine, caprine, camel, lama, equine, canine, feline, human, mice and chicken cytokine was 95, 95, 86, 83, 77, 75, 75, 61, 44 and 35%, respectively. Invitro expression of recombinant bovine IFN-gamma [rBoIFN-gamma] and secretion to culture medium was confirmed by ELISA test. Maximum expression of rBoIFN-gamma occurred at 96 and 144 h after transfection in COS-7 cells. These results showed that pcDNA3.1 expression vector and COS-7 cells transfected by diethylaminoethyl [DEAE]-dextran allowed the high level expression of bovine IFN-gamma gene and the release of protein in supernatant of cell culture

[ study of the genotypic variability at the MHC [B - F] in arian broiler chickens by PCR-SSCP]

The extreme polymorphism in MHC B-F genes enables the chickens to recognize enormous numbers of foreign peptides to trigger an immune reaction. Study of these genetic resources is very important and it is essential to avoid the loss of genetic variability. Genetic polymorphism among Arian strain, a endogenous broiler chickens in Iran, was investigated. In this study PCR-SSCP was carried out for revising the polymorphism in MHC [B-F] in Arian broiler chicken. 65 samples of chicken DNA belonged to 2 groups of parents [25] and hybrids [40] were analyzed. 7 distinct profiles have been demonstrated for two groups. Two profiles were identical between two groups. Two of all profiles have been demonstrated by previous studies and 5 were new in this study. From this study it was concluded that MHC [B-F] diversity exists within Arian broiler chicken that might be useful for selecting desirable immunological traits

Typing of Ovar-DRB1 second exon with PCR-RFLP technique in Iranian Shaul Sheep

Several studies have focused on polymorphisms of major histocompatibility complex [MHC] in sheep, Ovar-MHC. This molecule plays a pivotal role in antigen presentation for eliciting immune responses against invading pathogens. The best-characterized genetic control of disease resistance and immune response in animals is associated with MHC. Numerous molecular genetic investigations have been undertaken to detect polymorphisms of MHC genes and their association with resistance to infectious diseases. We have examined Ovar-DRB1 in DNA samples of 82 Shaul Sheep using polymerase chain reaction [PCR] and restriction fragment length polymorphism [RFLP] method. Identities of 8 different patterns and 5 distinct DRB1 alleles among Iranian Shaul Sheep have been determined. PCR-RFLP analysis allows rapid identification of Ovar-DRB1 types and enables rapid discrimination between homozygotes and heterozygotes. Data obtained from the present study have revealed that the exon 2 region of Ovar-DRB1 was highly polymorphic in sheep. So PCR-RFLP can be applied to the analysis of this locus

[Semiquantitative RT-PCR analysis to assess the expression levels of iNOS in chicken macrophages]

iNOS is inducible by a variety of factors related to inflammation and referred to as inducible NOS[iNOS]. It is regulated at the level of gene expression; once expressed, it produces NO at a high rate. iNOS gene-expression profiling is an important tool in understanding molecular markers of the responses of cells and tissues to external factors. In this article a semiquantitative reverse transcription-polymerase chain reaction [RT-PCR] protocol was optimized to extract RNA [ribonucleic acid] from chicken spleen and to measure the expression levels of iNOS mRNAs from each sample. Detailed procedure was described for the analysis of iNOS levels. beta-actin was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. Co-amplification of the iNOS gene with housekeeping gene [beta-actin] provides a quantitative result. Changes in gene expression level may be monitored, while avoiding sample-to-sample loading variation

RNA extraction and leptin receptor mRNA detection in bull ejaculated spermatozoa

Leptin, known as a potential satiety factor, plays an important role in both metabolism and reproduction. The presence of leptin in human seminal plasma and human spermatozoa has been shown; recently, leptin receptors [Ob-R] have been localized in human spermatozoa, thus suggesting a possible action of this hormone even on these cells. Our aim was to detect leptin receptor mRNA in bull ejaculated spermatozoa by reverse transcriptase-polymerase chain reaction [RT-PCR]. Total RNA was isolated from bull ejaculated spermatozoa and purified by different methods. Our results have revealed that sodium dodecylsulphate [SDS] and SDS/citric acid extraction methods are superior to guanidinium isothiocyanate in terms of yield and reproducibility of RNA recovery. The mRNA for Ob-Rb was detected in all samples examined. We conclude that Ob-R mRNA is present in bull spermatozoa where seminal plasma leptin can exert its effects

Molecular epidemiology of Salmonella Abortusovis in Iran

Study of different genotypes amongS.abortusovis strains. Observation study. 58 Salmonella abortusovis strains belonged todifferent countries were studied. IS200 fingerprinting by Southern blothybridisation have been applied for genotyping. 8 different genotypes identified within strainsunder study. All genotypes contained 3 to 5 copies ofIS200 and revealed high relatedness to their origins. Using IS200 fingerprinting for IranianS.abortusovis strains showed high polymorphism becauseeach province at least had one distinct pattern. Also thismethod remains as a sensitive method for typing theS.abortusovis

[Study of Salmonella plasmid virulence genes [spv] in Salmonella enterica serovars isolated in Iran]

Objective: to study the presence of spv genes among different Salmonella serovars that isolated in veterinary microbiology department

Design: observation study

Samples: a total of 138 Salmonella strains belonged to 9 different serovars were studied

Procedure: in this study we applied PCR method using PG44 and PG48 primers to amplify spvR gene in different serotypes

Results: in PCR amplification, serotyps Sabortusovis, S.dublin, S.typhimurium and S.brandburg developed the 890 bp amplicons. S.typhi, S.senfrenberg and S.bovismorbificans have yielded nonspecific bands of different sizes. S.newport revealed no band in amplification

Conclusion: salmonella serotypes such as typhi, senfrenberg and bovismorbiJicans with nonspecific bands in PCR amplification does not share virulence plasmids. Furthermore, spvR loci could be considered as a good marker for presence or absence of virulence plasmids in different Salmonella serotypes