Identification of nucleotide variation of growth hormone gene in rabbit populations reared in Bulgaria

[EN] Five rabbit populations of New Zealand White (NZW), Californian (CAL), crossbred NZW×GW and two generations of the synthetic population – SPF1 and SPF2 reared in Bulgaria were included in the present study with the aim of detecting the genetic variability of the growth hormone encoding gene (GH) via polymerase chain reaction with the restriction fragment length polymorphism analysis and direct sequencing. The targeted region of the rabbit GH gene was amplified and a fragment of a total of 231 bp was obtained in all studied populations. Allele identification was determined after enzymatic digestion, where two fragments of 62 and 169 bp correspond to allele C and an undigested fragment of 231 bp corresponds to allele T. Two additional bands of 107 and 124 bp evidenced A/G genetic polymorphism in the rabbit GH gene. Thirtyeight percent of the studied rabbits were carriers of the double mutation (C/T+A/G) in the same locus as the studied GH gene. The sequence analysis revealed two nucleotide substitutions – g.111C>T and g.156A>G in the non-coding region between the regulatory TATA box and 5’ UTR region, and a novel g.255G>A genetic variant in intron 1 of GH gene. The A>G transition was most frequent (40.57%), compared to the other ones, G>A (28.57%) and C>T (10.80%), respectively. The most frequent genotype in the NZW population was homozygous TT (0.93), with a prevalence of the T allele (0.97) over allele C (0.03) for g.111C>T SNP site. The distribution of the allele and genotype frequencies at the sites g.156A>G and g.255G>A in this rabbit group was identical, with the highest value of 0.93 for alleles A and G, respectively. The rabbit populations CAL and NZW×GW showed equal frequencies of the prevalent T allele (0.83) and for homozygous TT genotype (0.67) according to g.111C>T SNP. The highest values were obtained for the allele А (0.83) and for homozygous AA genotype (0.67) at c.33A>G SNP in these rabbit groups. The highest values (0.67, 0.60 and 0.80) for the heterozygous genotypes at g.111C>T, g.156A>G and g.255G>A SNPs, respectively, were detected among the SPF2 rabbit population, compared to the both homozygous genotypes. The results obtained in the present research indicates a significant degree of genetic variability of the studied polymorphic GH locus in the SPF2 rabbit group.Gencheva, DG.; Koynarski, TV.; Dafova, V.; Tanchev, SG. (2021). Identification of nucleotide variation of growth hormone gene in rabbit populations reared in Bulgaria. World Rabbit Science. 29(1):19-29. https://doi.org/10.4995/wrs.2021.12693OJS1929291Abdel-Kafy E., Hussein B., Abdel-Ghany S., El-Din A., Badawi Y. 2015. Single nucleotide polymorphisms in growth hormone gene are associated with some performance traits in rabbit. Int. J. Biol. Pharm. Allied Sci., 4: 490-504.Abdel-Kafy, E., Darwish, S., Elkhishin, D. 2016. Correlating single nucleotide polymorphisms in the myostatin gene with performance traits in rabbit. World Rabbit Sci., 24: 213-221. https://doi.org/10.4995/wrs.2016.4026Amalianingsih T., Brahmantiyo B. 2014. The variability of growth hormone gene associated with ultrasound imaging of longissimus dorsi muscle and perirenal fat in rabbits. Media Peternakan, 37: 1-7. https://doi.org/10.5398/medpet.2014.37.1.1Dimitrova I., Dimitrov T., Teneva A., Tzvetkova H. 2008. Rabbit production in Bulgaria. Biotechnol. Anim. Husbandry, 24: 149-154. https://doi.org/10.2298/BAH0802149DEl-Sabrout K., Aggag S. A. 2017. Associations between single nucleotide polymorphisms in multiple candidate genes and body weight in rabbits. Vet. World, 10: 136. https://doi.org/10.14202/vetworld.2017.136-139El-Sabrout K., Aggag S., de Souza Jr J.B.F. 2019. Some recent applications of rabbit biotechnology - a review. Anim. Biotechnol., 1-5. https://doi.org/10.1080/10495398.2018.1539005Fontanesi L., Dall'Olio S., Spaccapaniccia E., Scotti E., Fornasini D., Frabetti A., Russo V. 2012. A single nucleotide polymorphism in the rabbit growth hormone (GH1) gene is associated with market weight in a commercial rabbit population. Livest. Sci., 147: 84-88. https://doi.org/10.1016/j.livsci.2012.04.006Fontanesi L., Tazzoli M., Scotti E., Russo V. 2008. Analysis of candidate genes for meat production traits in domestic rabbit breeds. In Proc.: 9th World Rabbit Congress, June 10-13, 2008, Verona, Italy, 79-84.Gencheva D., Georgieva S., Velikov K., Koynarski T., Tanchev S. 2017. Single nucleotide polymorphism of the Growth Hormone Receptor (GHR ) encoding gene in Oryctolagus cuniculus. J. BioSci. Biotechnol., 6: 197-201.Heracle BioSoft. 2013. DNA Sequence Assembler v.4. https://www.DnaBaser.comHristova D.G., Tanchev S.G., Velikov K.P., Gonchev P.G., Georgieva S.J. 2018. Single nucleotide polymorphism of the growth hormone (GH ) encoding gene in inbred and outbred domestic rabbits. World Rabbit Sci., 26: 49-55. https://doi.org/10.4995/wrs.2018.7211Hristova D., Tanchev S., Velikov K., Gonchev P., Georgieva S. 2017. Rabbit growth hormone and myostatin gene polymorphisms. J. Agr. Res., 2: 000133. https://doi.org/10.23880/OAJAR-16000133Hussein B., Abdel-Kafy E.M., Abdel-Ghany S.M., Gamal A.Y., Badawi Y.M. 2015. Single nucleotide polymorphism in growth hormone gene are associated with some performance traits in rabbit. Int. J. Biol. Pharm. Allied Sci., 4: 490-504.Kumar S., Stecher G., Tamura K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol., 33: 1870-1874. https://doi.org/10.1093/molbev/msw054Migdal L., Palka S., Kmiecik M., Derewicka O. 2019. Association of polymorphisms in the GH and GHR genes with growth and carcass traits in rabbits (Oryctolagus cuniculus). Czech J. Anim. Sci., 64: 255-264. https://doi.org/10.17221/27/2019-CJASNei M. 1973. Analysis of gene diversity in subdivided populations. Proc. Nat. Acad. Sci. USA, 70: 3321-3323. https://doi.org/10.1073/pnas.70.12.3321Sahwan F.M., El-Sheik, A.I., Sharaf, M.M., El-Nahas, A.F. 2014. Genetic polymorphism in growth hormone receptor gene (GHR) and its relationship with growth trait in pure and hybrid rabbit breeds. Alexandria J. Vet. Sci., 43: 45-51. https://doi.org/10.5455/ajvs.165197Stothard P. 2006. Sequence Extractor. https://www.bioinformatics. org/seqext/Tamura K., Nei M. 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol., 10: 512-526. https://doi.org/10.1093/oxfordjournals.molbev.a040023Thompson J.D., Higgins D.G., Gibson T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22: 4673-4680. https://doi.org/10.1093/nar/22.22.4673Wallis C., Wallis M. 1995. Cloning and characterisation of the rabbit growth hormone-encoding gene. Gene, 163: 253-256. https://doi.org/10.1016/0378-1119(95)00429-AYeh F., Yong R. 1999. POPGENE version 1.31 (02.04.2011). Microsoft based Freeware for Population Genetic Analysis. University of Alberta, Edmonton, Canada, http://www.ualberta.ca/~fyeh/fyehZaghloul A. R.; Khalil, M.H.; Iraqi, M. M.; Ramadan, Sh. and EL Nagar, A. G. 2019. Crossbreeding effects and polymorphic associations of genotypes of GH gene with growth traits in rabbits. Egyptian Journal of Rabbit Science, 29: 149-169. https://doi.org/10.21608/ejrs.2019.81100Zhang W.X., Zhang G.W., Peng J., Lai S.J. 2012. The polymorphism of GHR gene associated with the growth and carcass traits in three rabbit breeds. In Proc.: 10th World Rabbit Congress, Sharm El-Sheikh, Egypt, 75-78

Single nucleotide polymorphism of the Growth Hormone Receptor (GHR) encoding gene in Oryctolagus cuniculus

Considered the rabbit Growth Hormone Receptor (GHR) gene as a candidate gene for growth efficiency, understanding the genetic variation in this locus is of particular relevance. The aim of the present research was to investigate populations from the rabbits (Oryctolagus cuniculus) and to identify single nucleotide polymorphism (SNP) with respect to the GHR gene through PCR-RFLP assay. Genotype profiles were established in a total of 100 rabbits from two populations (New Zealand White, NZW, n=51 and Californian, n=49), reared at The Institute of Animal Science, Kostinbrod. As expected, a 479bp amplicon of the polymorphic site (exon 3) of GHR gene was amplified using PCR and digested with endonuclease enzymes HinfI. The restriction pattern obtained in agarose gel electrophoresis was constituted by three bands (210bp, 162bp and 107 bp) for allele C and by two bands for allele G (317bp and 162 bp). The obtained restriction fragments revealed three genotypes: CC, CG and GG, observed in 7.8%, 53% and 39.2% of the NZW rabbit population and in 28.6%, 51% and 20.4% of the Californian rabbit population, respectively, without departure from the Hardy-Weinberg equilibrium (P>0.20) in the investigated groups. The allele frequencies determined a prevalence of the G allele (0.657) over the C allele (0. 343) in NZW rabbit population, while in the Californian rabbits, the frequency of the C allele (0.541) was higher than allele G (0.459). Observed heterozygosity was higher than expected, resulting in a negative inbreeding coefficient (Fis= -0.174 for NZW rabbits and Fis= -0.027 for Californian rabbits), indicating a sufficient number of heterozygous forms in both studied groups of rabbits. The obtained results from the present investigation confirmed the presence of the SNP in rabbit GHR gene. Therefore, the genetic variability established in this polymorphic locus could be applied in further association studies with growth traits in domestic rabbits

Влияние на силимарин и охратоксин А върху хуморалния естествен имунитет при пилета бройлери

The aim of this work was to investigate the effect of Ochratoxin A (OTA) and Silymarin on serum lysozyme concentrations, complement and betalysin activity in broiler chickens. In this experiment 144 one-day-old Ross 308 male broiler chicks were used. All chicks were divided in four groups of 36 birds each: Group 1: Basal diet (BD) with no supplementation of Ochratoxin A (OTA) and Silymarin; Group 2: BD with 1.0% Silymarin; Group 3: BD with 3.0 mg/kg OTA; Group 4: BD with 3.0 mg/kg OTA plus 1.0% Silymarin. It was found that lysozyme concentration in the 2nd group there is a significant difference to groups 3 and 4. The immunosuppressive effect of Ochratoxin A is underlined but no protective effect of Silymarin in the group 4 was found. The alternative pathway of complement activation (APCA) is affected in the group 4. Betalysine there is a significant decreasing in the group 3 but slightly increasing of Betalysine in 4th group. Based on these results it can be concluded that OTA there is an immunosuppressive effect on the studied traits and there is a positive effect of Silymarin only on serum betalysineЦелта на това изследване е да се проучи влиянието на Охратоксин А (ОТА) и Силимарин върху концентрациите на серумния лизоцим, активността на комплемента и бетализина при пилета бройлери. За този експеримент бяха използвани 144 еднодневни мъжки пилета бройлери от хибрида Ross 308. Пилетата бяха разпределени в четири групи по 36 пилета: Първа група (1): Основна диета (ОД) без добавяне на ОТА и Силимарин; Втора група (2): ОД с добавяне на 1.0% Силимарин; Трета група (3): ОД с добавяне на 3.0 mg/kg OTA; Четвърта група (4): ОД с добавяне на 3.0 mg/kg OTA и 1.0% Силимарин. Беше намерено, че лизоцимната концентрация при втора група е достоверно по-висока от тази при групи 3 и 4. Имуносупресивният ефект на Охратоксин A е подчертан, но не се установява протекция от Силимарина при 4-та група. Активността на алтернативния път за активиране на комплемента е понижена при 4-та група. Активността на бетализина е достоверно по-ниска при 3-та група, но е достоверно увеличена при 4-та група. Въз основа на получените резултати може да се заключи, че ОТА има подчертан имуносупресивен ефект върху изследваните показатели при пилетата, но Силимарина има известен положителен ефект само върху активността на бетализина

Adenovirus-vectored novel African Swine Fever Virus antigens elicit robust immune responses in swine

<div><p>African Swine Fever Virus (ASFV) is a high-consequence transboundary animal pathogen that often causes hemorrhagic disease in swine with a case fatality rate close to 100%. Lack of treatment or vaccine for the disease makes it imperative that safe and efficacious vaccines are developed to safeguard the swine industry. In this study, we evaluated the immunogenicity of seven adenovirus-vectored novel ASFV antigens, namely A151R, B119L, B602L, EP402RΔPRR, B438L, K205R and A104R. Immunization of commercial swine with a cocktail of the recombinant adenoviruses formulated in adjuvant primed strong ASFV antigen-specific IgG responses that underwent rapid recall upon boost. Notably, most vaccinees mounted robust IgG responses against all the antigens in the cocktail. Most importantly and relevant to vaccine development, the induced antibodies recognized viral proteins from Georgia 2007/1 ASFV-infected cells by IFA and by western blot analysis. The recombinant adenovirus cocktail also induced ASFV-specific IFN-γ-secreting cells that were recalled upon boosting. Evaluation of local and systemic effects of the recombinant adenovirus cocktail post-priming and post-boosting in the immunized animals showed that the immunogen was well tolerated and no serious negative effects were observed. Taken together, these outcomes showed that the adenovirus-vectored novel ASFV antigen cocktail was capable of safely inducing strong antibody and IFN-γ⁺ cell responses in commercial swine. The data will be used for selection of antigens for inclusion in a multi-antigen prototype vaccine to be evaluated for protective efficacy.</p></div

Application of the Natural Products NOZEMAT HERB and NOZEMAT HERB PLUS Can Decrease Honey Bee Colonies Losses during the Winter

Honey bees (Apis mellifera L.) are crucial pollinators for many crops and natural ecosystems. However, honey bee colonies have been experiencing heavy overwinter mortality in almost all parts of the world. In the present study we have investigatеd, for the first time, the effects from the application of the herbal supplements NOZEMAT HERB® (NH) and NOZEMAT HERB PLUS® (NHP) on overwintering honey bee colony survival and on total protein and lysozyme content. To achieve this, in early autumn 2019, 45 colonies were selected and treated with these herbal supplements. The total protein and lysozyme content were evaluated after administration of NH and NHP twice the following year (June and September 2020). The obtained results have shown that both supplements have a positive effect on overwintering colony survival. Considerable enhancement in longevity of “winter bees” has been observed after the application of NHP, possibly due to the increased functionality of the immune system and antioxidant detoxification capacity. Although the mechanisms of action of NH and NHP are yet to be completely elucidated, our results suggest a new holistic approach on overwintering honey bee colony survival and welfare

Practical Application and Influence of “Avigen duck” Immunomodulator to White Pekin Ducks Humoral Immune Factors

Factors of non-specific immunity are responsible for protecting birds from a number of viruses and bacteria. Bactericidal proteins are played here lysozyme and complement, interferon, as well as beta-lysine. Adding appropriate active compounds to the bird's diet to activate the innate immune response are subject to studies by many authors. The practical application and influence of the "Avigen Duck". immunomodulator in the cultivation of White Pekin ducks aims to clarify the dynamics of the indicators. It was found that the ducks treated with the immunomodulator had higher non-specific protection expressed in the values of the observed indicators. Complement activity for the experienced group (529.45 ± 17,85 CH50) is significantly higher than that of untrained birds (308.56 ± 10.19 CH50), on the 30th day of their lives (P <0.001). Lysozyme values for the experienced group (6.34 ± 0.86 mg L-1) is more than twice as high as its concentration in control birds - 2.52 ± 0.59 mg L-1. At 30 days of age, the mean concentration of IFN γ in the control group of ducks was 108.86±6.12 pg ml-1, and in the experimental group treated with immunomodulator - 518.06±12.80 pg ml-1, accompanied by an increase in the values of IL-2 and IL-6 and beta-lysine activity. These data show that, despite the short life of the ducks, the concentrations of the investigated factors of non-specific immunity increase significantly and statistically reliably, even after a single treatment with immunomodulator "Avigen Duck". The practical application of the immunomodulator in the duck diet has a strong effect on their non-specific immunity

Development and Optimization of an Enzyme Immunoassay to Detect Serum Antibodies against the Hepatitis E Virus in Pigs, Using Plant-Derived ORF2 Recombinant Protein

Hepatitis E is an emerging global disease, mainly transmitted via the fecal–oral route in developing countries, and in a zoonotic manner in the developed world. Pigs and wild boar constitute the primary Hepatitis E virus (HEV) zoonotic reservoir. Consumption of undercooked animal meat or direct contact with infected animals is the most common source of HEV infection in European countries. The purpose of this study is to develop an enzyme immunoassay (EIA) for the detection of anti-hepatitis E virus IgG in pig serum, using plant-produced recombinant HEV-3 ORF2 as an antigenic coating protein, and also to evaluate the sensitivity and specificity of this assay. A recombinant HEV-3 ORF2 110-610_6his capsid protein, transiently expressed by pEff vector in Nicotiana benthamiana plants was used to develop an in-house HEV EIA. The plant-derived HEV-3 ORF2 110-610_6his protein proved to be antigenically similar to the HEV ORF2 capsid protein and it can self-assemble into heterogeneous particulate structures. The optimal conditions for the in-house EIA (iEIA) were determined as follows: HEV-3 ORF2 110-610_6his antigen concentration (4 µg/mL), serum dilution (1:50), 3% BSA as a blocking agent, and secondary antibody dilution (1:20 000). The iEIA developed for this study showed a sensitivity of 97.1% (95% Cl: 89.9–99.65) and a specificity of 98.6% (95% Cl: 92.5–99.96) with a Youden index of 0.9571. A comparison between our iEIA and a commercial assay (PrioCHECK™ Porcine HEV Ab ELISA Kit, ThermoFisher Scientific, MA, USA) showed 97.8% agreement with a kappa index of 0.9399. The plant-based HEV-3 ORF2 iEIA assay was able to detect anti-HEV IgG in pig serum with a very good agreement compared to the commercially available kit