Ovine Serum Research Articles

The effect of ovine, bovine and human umbilical cord blood sera on in vitro maturation of sheep oocytes

The aim of the present study was to evaluate the effect of ovine umbilical cord serum (OUCS), bovine umbilical cord serum (BUCS) and human umbilical cord serum (HUCS) on in vitro maturation, cleavage and developmental capacity of sheep oocytes. Oocytes were treated in maturation media with different concentration (10 and 20%) of OUCS, BUCD, HUCS and FCS to examine effects on early embryonic development. The processing results showed that the addition of 10% OUCS (27.1%) and BUCS (24.8%) can improve (P<0.05) the mean blastocyst formation compared maturation medium with FCS (17.1%). However, there was no significant difference (P>0.05) in blastocyst rate between OUCS (20.2%), BUCS (21.8%), HUCS (18.2%) and FCS (23.2%) when sera concentrations were increased from 10 to 20%. In conclusion, OUCS and BUCS alone (without growth factor supplement) could improve the embryo production and early embryonic development of sheep oocytes. However, the positive effects of umbilical cord sera on embryonic development may remove by high concentrations of umbilical cord sera.

Seropositivity to Toxoplasma infection in sheep samples submitted to Animal and Plant Health Agency laboratories between 2005 and 2012

Ovine serum samples submitted to Animal and Plant Health Agency (APHA) (formerly the Animal Health and Veterinary Laboratories Agency) – Weybridge regional laboratories in England and Wales for diagnostic and...

Determination of the glass-transition temperature of proteins from a viscometric approach

All fully hydrated proteins undergo a distinct change in their dynamical properties at glass-transition temperature Tg. To determine indirectly this temperature for dry albumins, the viscosity measurements of aqueous solutions of human, equine, ovine, porcine and rabbit serum albumin have been conducted at a wide range of concentrations and at temperatures ranging from 278K to 318K. Viscosity–temperature dependence of the solutions is discussed on the basis of the three parameters equation resulting from Avramov's model. One of the parameter in the Avramov's equation is the glass-transition temperature. For all studied albumins, Tg of a solution monotonically increases with increasing concentration. The glass-transition temperature of a solution depends both on Tg for a dissolved dry protein Tg,p and water Tg,w. To obtain Tg,p for each studied albumin the modified Gordon–Taylor equation was applied. This equation describes the dependence of Tg of a solution on concentration, and Tg,p and a parameter depending on the strength of the protein–solvent interaction are the fitting parameters. Thus determined the glass-transition temperature for the studied dry albumins is in the range (215.4–245.5)K.

ChemInform Abstract: Knoevenagel Condensation of Diethylmalonate with Aldehydes Catalyzed by Immobilized Bovine Serum Albumin (BSA).

AbstractBovine serum albumin immobilized covalently on an epoxy‐functionalized polymeric support is developed and utilized as catalyst for the condensation of aliphatic and aromatic aldehydes with diethyl malonate.

Rhipicephalus (Boophilus) microplus tick in vitro feeding methods for functional (dsRNA) and vaccine candidate (antibody) screening

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5–6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated (CPDA) and EDTA tubes. Although >30mg semi-engorged ticks fed more reliably, ticks as small as 15mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86 did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA. This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and treatments.

Thermoresponsive hydrogels from BSA esterified with low molecular weight PEG

ABSTRACTBovine serum albumin (BSA) and low molecular weight polyethylene glycol (PEG) were reacted in a single‐step reaction to synthesize translucent hydrogels with a sol–gel transition at temperatures between 37 and 40°C. Gelation occurred by aggregation of smaller assemblies of BSA–PEG precursors within minutes. The sol–gel transition concentration depended on the molecular weight of PEG only at temperatures below 35°C; above 45°C phase separation occurred and a precipitate formed. Microscopic examination showed the porous structure of the gels. At a fairly low grafting ratio, BSA preserved its native secondary and tertiary structure and maintained its capability for binding and enclosing small molecules. Drug delivery was assessed by a discontinuous method in vitro using 5‐fluorouracil. Degradation tests with trypsin confirmed that the hydrogels were biodegradable. This novel material holds promise for biomedical applications as potentially injectable drug delivery vehicle. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40946.

Kinetics and efficiency of a methyl-carboxylated 5-Fluorouracil-bovine serum albumin adduct for targeted delivery.

5-Fluorouracil (5-FU) is a clinically well-established anti-cancer drug effectively applied in chemotherapy, mainly for the treatment of breast and colorectal cancer. Substantial disadvantages are adverse effects, arising from serious damage of healthy tissues, and shortcoming pharmacokinetics due to its low molecular weight. A promising approach for improvement of such drugs is their coupling to suitable carriers. Here, a 5-FU adduct, 5-fluorouracil acetate (FUAc) is synthesized and covalently coupled to bovine serum albumin (BSA) as model carrier molecule. On average, 12 molecules FUAc are bound to one BSA. Circular dichriosm (CD)-spectra of BSA and FUAc-BSA are identical, suggesting no significant conformational differences. FUAc-BSA is tested on T-47D and MDA-MB-231 breast cancer cells. Proliferation inhibition of membrane albumin-binding protein (mABP)-expressing T-47D cells by FUAc-BSA is similar to that of 5-FU and only moderate for MDA-MB-231 cells that lack such expression. Therefore, a crucial role of mABP expression in effective cell growth inhibition by FUAc-BSA is assumed.

Prevalence of antibodies against Parainfluenza virus type 3, Respiratory syncitial virus and bovine Herpesvirus type 1 in sheep from Northern Prefectures of Japan.

Ovine sera collected in the Prefectures of Hokkaido, Aomori and Iwate in the Northern Japan were examined for the presence of antibodies against Respiratory syncytial virus (RSV), bovine Herpesvirus type 1 (infectious bovine rhinotracheitis: IBR) and Parainfluenza virus type 3 (PIV3) using serum neutralisation (SN) and enzyme-linked immunosorbent assay (ELISA) tests. Twenty-three animals (11.73%) out of the 196 tested were sero-positive to PIV3. Sixteen animals (8.69%) out of the 184 tested reacted to RSV. No animals were positive to IBR antigen. Sero-conversions to PIV3 were detected in Hokkaido and Iwate (14.92% and 8.82%, respectively). Antibodies against RSV were detected in Hokkaido (9.23%) and Aomori (14.28%). Although no diagnostic measures were in place, the infections did not appear to be related to any reduction in sheep productivity.

Prevalence and risk factors associated to ovine toxoplasmosis in northeastern Brazil

In this study, we aimed to determine the prevalence and risk factors associated to Toxoplasma gondii infection in sheep from northeastern Brazil. A total of 932 ovine serum samples from 54 properties in 19 municipalities of the state of Sergipe were collected and assayed using indirect fluorescent antibody test. The assay used antibodies against Toxoplasma gondii (IFAT-IgG) with a cutoff point of 1:64. We observed that 28.22% (263/932) of the ovine samples were serum-reactive. In a logistic regression, factors such as consumption of water directly from the source, consumption of water from a deep well, and age below 12 months were associated with protection; whereas factors such as presence of cats on the property, presence of slatted floor, and use of exchanged or borrowed breeding males were associated with infection. The studied area can be considered endemic for toxoplasmosis, so it is necessary to adopt preventive and control measures because this zoonotic infection poses risks to public health.

Orally administered ovine serum immunoglobulins modulate the intestinal levels of Lactobacillus and enterobacteria in the growing rat1

The aim was to determine whether orally administered ovine serum immunoglobulins modulate the gut microbiota in the growing rat. Thirty Sprague-Dawley male rats were used in a 21-d study and fed either a basal control diet (control; no immunoglobulin) or a similar diet containing freeze-dried ovine immunoglobulin (ovine Ig) with 15 individually fed rats per diet. Bacterial DNA isolated from ileal and colonic digesta were subjected to PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In the ileum, the DGGE band number and diversity index were greater (P < 0.05) for rats fed the ovine Ig than those fed the control diet. The DNA sequencing of a selected DGGE band in the ovine Ig-fed rats revealed 99% similarity to the Lactobacillus strains. The quantitative PCR data revealed that supplementation of the diet with the ovine Ig fraction supported the growth of Lactobacillus and conversely decreased the number of enterobacteria in ileal and colonic digesta. Inclusion of the ovine Ig fraction led to a greater (P < 0.05) ratio for total Lactobacillus to total bacteria and total Lactobacillus to enterobacteria. The results from the present study show that dietary supplementation with ovine Ig may alter the intestinal environment by a specific enrichment of Lactobacillus strains and depletion of enterobacteria.

Changes in blood flow in ovine follicles and serum concentration of estradiol 17 beta (E2) and nitric oxide (NO) around the time of ovulation in Ossimi ewes

The aim of the present study was to examine the relation between follicular blood flow of the ovulatory follicle and the levels of serum E2 and nitric oxide (NO) in Ossimi ewe. Seven cyclic ewes were synchronized with a double injection PGF2α. The follicular wave was examined daily until ovulation (disappearance of the large dominant follicle ultrasonographically) with transrectal color Doppler ultrasonography (8–10MHz linear array transducer). The number of recruited follicles was 4.8±0.9 (3–8 follicles) with diameter of 2.8±0.1mm. The interval from PGF2α injection to follicle deviation was 2.35±0.07 days. The diameter of the first largest follicle (LF1) at recruitment day was 4±0.3mm while the diameter of the second largest follicle (LF2) was 3.7±0.1mm. The diameter of LF1 at the day of deviation was 5.1±0.5mm while the diameter of the LF2 was 4±0.7mm. The diameter of the ovulatory follicle was 6.1±0.5at day of ovulation. We detected the blood flow area of the ovulatory follicle at D2. At ovulation, the blood flow area and blood flow area percent increased significantly to be 11.9±0.6mm2 and 44±3.4% respectively. The results showed a positive correlation between E2 and NO (r=0.85, P<0.009). Both increased concomitantly with the diameter of the ovulatory follicle. Besides, NO and E2 reached a maximum level at ovulation (12.1±1.8ng/ml and 16.4±1.7pg/ml respectively).

Recovery of intact IgG in the gastrointestinal tract of the growing rat following ingestion of an ovine serum immunoglobulin

The aim of this study was to determine whether orally ingested ovine serum IgG partly resists digestion in the growing rat. Fifteen Sprague-Dawley male rats were allocated to one of three diets for a 3-week study: a control diet (CON) and two test diets containing either freeze-dried ovine serum immunoglobulin (FDOI) or inactivated ovine serum immunoglobulin (IOI). Samples of stomach chyme and intestinal digesta from the ad libitum-fed rats were subjected to ELISA and Western blot analysis. Amounts of intact ovine IgG for the FDOI diet were found to be 13.9, 20.0, 34.1, 13.0 and 36.9 μg in the total wet digesta from the stomach chyme, duodenal, jejunal, ileal and colonic digesta respectively. Qualitative detection by Western blot revealed the presence of intact ovine serum IgG with a ~150 kDa MW. This was detected in all of the gut segments (stomach chyme, duodenal, jejunal, ileal and colonic digesta) for growing rats fed the FDOI diet. No ovine IgG was detected in the chyme or digesta from rats fed the CON or the IOI diets. Ovine serum IgG partly resisted digestion in the growing rat fed the FDOI diet and was found throughout the digestive tract. These results provide a basis to explain the reported biological effects of orally administered immunoglobulin.

Intact but not denatured ovine serum immunoglobulins positively modulate mucosal immune mediators in the growing rat challenged with Salmonella enteritidis

Immunoglobulins are major glycoproteins that modulate the immune response of gut-associated lymphoid tissue. In the present study, we sought to determine whether orally administered ovine serum immunoglobulins modulate selected indices of mucosal immune function and immune mediators in the growing rat challenged with Salmonella enteritidis. Rats were fed a casein-based basal control diet (BD; unchallenged). Three groups of rats were challenged orally with 1 x 10(7) viable S. enteritidis on day 15 of the study and were fed the BD, the BD containing freeze-dried ovine immunoglobulins (FDOI), or the BD containing autoclaved ovine immunoglobulins (AOI; negative control diet). The rats were randomly allocated to one of the four groups (n 15) and consumed their diet for 18 d. In all of the intestinal segments, the challenged rats fed either the BD or AOI diet produced higher (P<0·05) mucosal levels of interferon-g, TNFa, IgA and myeloperoxidase activity than the challenged rats fed the FDOI diet. In contrast, IL-4 and IL-10 levels were higher in the challenged FDOI-fed rats compared with the other challenged groups. The challenged FDOI-fed rats had higher (P<0·05) mucosal anti-Salmonella IgA and IgG in all of the intestinal segments except the jejunum and ileum. Generally, the challenged rats receiving the FDOI diet had significantly (P<0·05) higher mucosal mucin protein content compared with challenged rats receiving either the BD or AOI diet. In conclusion, an ovine immunoglobulin fraction positively modulated some selected indices of mucosal immune function and its mediators in growing rats challenged with S. enteritidis.

Self‐assembly of bovine serum albumin and poly(acrylic acid) induced by noncovalent bonds

AbstractBovine serum albumin/poly(acrylic acid), BSA/PAA, nano‐scaled particles were produced by noncovalent bonds induced self‐assembly method at acid pH area. Proper conditions during preparation process, such as pH value, BSA/PAA weight ratio(WR), PAA molecular weight, were researched by studying the hydrodynamic diameter, polydispersity index, and ζ potential of the nanoparticles. Complex formation between BSA and PAA was studied by FT‐IR, AFM, and TEM. BSA chains are supposed to be partly trapped in the nanoparticles core after interaction with PAA because of the electrostatic attractions and hydrogen bonds interactions between BSA and PAA, while the rest of the BSA chains should form the shell of the nanoparticles. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

A serologicalsurveillance of bluetongue disease in sheep and goats in Iraq by using acompetitive ELISA Technique

In this study, a sero-survillance on ovine and caprine serum samples from all over Iraqexcept the governorates of Iraqi Kurdistan region was conducted. It aimed for the detection ofspecific antibodies directed against VP7 protein - the sero-group specific antigen of bluetongue virus – by using a competitive ELISA technique.The results showed that among a totalof 3277 serum samples, 1436 samples were found to be positive forming 43.82% of totalsamples, while 1687 samples were negative with 51.48 % and 154 sample were suspiciousforming 4.69 %. The highest positive serum samples were found in Al Basrah Province(southern of Iraq) with 60.41% and in Al Anbar Province (Western of Iraq) with 61%, whilethe lowest of positive samples were found in Nineva (Northern of Iraq) with 22.35%.The results also showed a closely ratio between positive percentage of goats and sheepsamples with the positive goats serum samples ( in five provinces ) were 45 of 114 samplesrepresenting 39.47 %,while the positive sheep serum samples ( in fifteen provinces) were1391 of 3163 samples representing43.97%. Our study is complementary to the previousworks held in this field and it represents the first field serological survey for bluetongueinsheep and goats in Iraq.

Expression of endogenous beta retroviruses and Hyal-2 mRNA in immune organs of fetuses and lambs

Endogenous beta retroviruses (enJSRV) are highly homologous with Jaagsiekte sheep retrovirus (exJSRV), this exogenous retrovirus is the aetiological agent of ovine pulmonary adenocarcinoma (OPA). The aim of this study was to clarify the function of enJSRV and the immunological mechanisms of its corresponding antibody, that is undetectable in JSRV-infected ovine serum. The expression of enJSRV envelope protein and Hyal-2 mRNA in immune organs and lungs of ovine fetuses and lambs were analyzed by Real-Time reverse transcription PCR and In Situ Hybridization using specific probes. In Situ Hybridization results indicated that the enJSRV envelope protein and Hyal-2 mRNA were expressed in thymus, spleen, mesenteric lymph nodes and lungs at different times, while no positive signals were detected in the negative controls. On the other hand, results from Real-Time reverse transcription PCR analysis showed that in 130d fetuses and 3d newborn lambs the enJSRV mRNA levels were much higher in organs associated with the immune system than that in lungs, especially in the thymus and spleen, but levels of Hyal-2 mRNA expression was not significantly different in all collected tissue. These results provided evidence from an immunology point of view to understand why the circulating antibodies against exJSRV are undetectable in JSRV-infected ovine, and will help to unravel the pathogenesis of JSRV-infected ovine.

Serological Survey to Determine the Occurrence of Blue Tongue Virus, Bovine Leukemia Virus and Herpesvirus Infections in the Japanese Small Ruminant Population from Northern Districts

Ovine sera, collected from the northern prefectures of Hokkaido, Aomori and Iwate in Japan, were examined for the presence of antibodies against Blue tongue virus (BTV), bovine leukemia virus (BLV), bovine Herpesvirus type 1, agent of infectious bovine rhinotracheitis (IBR), ovine Herpesvirus type 2 (OvHV-2), agent of sheep-associated malignant catarrhal fever (SA-MCF), and bovine Herpesvirus type 4 (BoHV-4), using agar immune diffusion, serum neutralisation (SN) and enzyme linked immunosorbent assay (ELISA) tests. No animals were positive to IBR, BoHV- 4 or BLV antigens. Antibodies against BTV have been detected in 3 samples (1.11%) in two flocks from Hokkaido. The seroprevalence of OvHV-2 was observed in twelve flocks from the 3 considered prefectures, in 56 sheep and two goats, with 37.66% of samples giving a positive reaction in the serum neutralization test. The infections did not appear to be related to the reduction in sheep productivity. Immune reaction reported in goats could refer to Caprine Herpesvirus-2 (CpHV-2). These results indicate that sheep are reservoirs for OvHV-2 in the field in Japan.

Rapid immunofiltration assay based on colloidal gold–protein G conjugate as an alternative screening test for bovine and ovine brucellosis

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle-protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar's analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.

Establishment and Characterization of Gene Expression of Two New Ovine Mammary Cell Lines: Preliminary Results.

Some post-translational modifications are necessary for the production of biopharmaceutical proteins, with a good specific action and a high biological activity. These modifications are obtained only by bioreactors based on eukaryotic cell as mammary cells. Bioreactors in vitro, with this kind of cell have been used for a viable and strategic production of biologically active recombinant proteins. For this reason, the establishment of a new line of mammary cells with high milk protein expression is an essential work. The main objectives of this study was to compare two methods, enzymatic and non-enzymatic, to establish ovine mammary cells culture and verify their gene expression of milk proteins such as beta lactoglobulin, alpha casein, beta casein and kappa casein with different treatments: LOS (lactating ovine serum) or FBS (fetal bovine serum) added to the culture medium, in the presence or absence of Matrigel. In this manner, an in vitro study was performed and the culture of two lines were established, digested (DL) and non-digested (NDL), of ovine mammary cell until the passage 12 (P12). In DL was observed just one cellular type that was positive for staining with vimentin. This cell line expressed beta lactoglobulin and beta casein genes with the FBS treatment and without Matrigel. The gene expression was lower (P=0.001) when compared to the NDL under the same conditions of culture. Then, the NDL expressed beta lactoglobulin, beta casein and kappa casein genes when treated with FBS without Matrigel. The treatment with LOS in the culture medium increased the gene expression of beta lactoglobulin for both cell lines. The growth curve was determined with both cell lines in P12 with FBS or LOS treatment. For the NDL, the type of medium had effect on the cell growth speed and was highest with the FBS treatment (P<0.05). However, the medium did not have effect on growth speed of LD (P>0.05) and no difference was observed at the NDL treated with LOS (P>0.05). The NDL was positive for staining with vimentin and cytokeratin. In conclusion, the in vitro culture of NDL and DL was established up to the P12 with expression of milk protein and the LOS treatment increased the expression of beta lactoglobulin. The two cell lines culture were positive for staining of vimentina but only NDL was positive for cytokeratin. Research supported by FAPESP (2008/56138-2) and CNPq. (poster)

Evaluation of an Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Antibodies against Anaplasma phagocytophilum in Sheep

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Anaplasma phagocytophilum in ovine serum samples was evaluated. The assay used purified A. phagocytophilum grown in tick cell cultures as antigen. Serum samples were diluted 1 in 200 and binding was detected with anti-sheep IgG conjugated to horseradish peroxidase. All tests were carried out in the presence of positive and negative control samples. Optical density (OD) values obtained for each test sample at 490 nm were used to calculate percentage positivity (PP) of each sample based on the ratio of the OD of the test sample that of the positive reference sample. Known negative samples (n=69) obtained from uninfected sheep bred and maintained in a tick-free environment and subsequently shown to be susceptible to A. phagocytophilum were used to establish the cut-off point between negative and positive samples and to establish the specificity of the test. Serum samples obtained from 92 animals 14-21 days after infection were used to establish the sensitivity of the test. Using a cut-off point of 20PP (mean+2 standard deviations of the PP of 69 control samples) the test was shown to have a sensitivity of 84.8% and a specificity of 95.7%. Lowering the cut-off point to 15PP increased the sensitivity to 94.6%, but reduced the specificity to 92.8%.