Bovine Sera Research Articles

Disulfide bond generation in mammalian blood serum: detection and purification of quiescin-sulfhydryl oxidase

A sensitive new plate-reader assay has been developed showing that adult mammalian blood serum contains circulating soluble sulfhydryl oxidase activity that can introduce disulfide bonds into reduced proteins with the reduction of oxygen to hydrogen peroxide. The activity was purified 5000-fold to >90% homogeneity from bovine serum and found by mass spectrometry to be consistent with the short isoform of quiescin-sulfhydryl oxidase 1 (QSOX1). This FAD-dependent enzyme is present at comparable activity levels in fetal and adult commercial bovine sera. Thus cell culture media that are routinely supplemented with either fetal or adult bovine sera will contain this facile catalyst of protein thiol oxidation. QSOX1 is present at approximately 25nM in pooled normal adult human serum. Examination of the unusual kinetics of QSOX1 toward cysteine and glutathione at low micromolar concentrations suggests that circulating QSOX1 is unlikely to significantly contribute to the oxidation of these monothiols in plasma. However, the ability of QSOX1 to rapidly oxidize conformationally mobile protein thiols suggests a possible contribution to the redox status of exofacial and soluble proteins in blood plasma. Recent proteomic studies showing that plasma QSOX1 can be utilized in the diagnosis of pancreatic cancer and acute decompensated heart failure, together with the overexpression of this secreted enzyme in a number of solid tumors, suggest that the robust QSOX assay developed here may be useful in the quantitation of enzyme levels in a wide range of biological fluids.

Autochthonous tick-borne encephalitis virus (TBEV) - seropositive cattle in Belgium: a risk-based targeted serological survey

Tick-borne encephalitis virus (TBEV) is the most important arthropod-borne virus in Europe. The Western subtype of this pathogenic neurotropic flavivirus is carried by Ixodes ricinus. Tick-borne encephalitis has become a considerable public risk in several European countries, with currently 3000 hospitalized cases per year. The risk of TBEV-introduction into Belgium remains high and the presence of infected wildlife in Belgium is suspected. Domestic animals such as dogs or cattle can serve as excellent sentinels for TBEV-surveillance in order to install an early warning surveillance component for this emerging zoonotic disease of public importance. In a targeted, risk-based and cross-sectional sampling design, serological screening was performed on Belgian cattle (n = 650), selected from the 2010 Belgian national cattle surveillance serum bank. The three most Eastern provinces of Belgium, which are geographically situated closest to known and/or recently emerging TBEV-endemic, were targeted. These areas are also currently known as endemic for Lyme disease (Borrelia burgdorferi), another disease transmitted by the same tick. Bovine sera were tested at the TBEV Belgian National Reference Centre at the WIV-ISP, by gold standard TBEV seroneutralisation test, based on the rapid fluorescent focus inhibition test (RFFIT) protocol. Using a conservative >1/15 cut-off titer for SN test, 17 bovines were seropositive and six had borderline results (1/10 < titer < 1/15). The accuracy of the RFFIT-SNT was confirmed in a mouse inoculation test and by West Nile virus and Rabies virus serology. There was a positive correlation between the neutralizing antibody titer, determined by SN, and the median survival time in mice inoculated intranasally with a mix of virus and serum. Lesions consistent of viral encephalitis were demonstrated in histopathology. The overall bovine TBEV-seroprevalence in the targeted area was estimated between 2.6 and 4.3% and freedom could no longer be substantiated. Bovines with borderline results were often located close to confirmed seropositive animals. The geographical locations roughly coincided with the known Belgian hot spots for Lyme disease. This risk-based cross-sectional serological survey in cattle, obtained through one health cooperation confirms the presence of infected foci in Belgium for the first time. Given the relevance of TBEV for the food chain through consumption of unpasteurized milk and cheese and through its considerable public burden in other European countries, further surveillance in cattle, other sentinels, ticks and humans at risk is recommended to further determine the location and size of endemic foci and the risk for public health.

Comparison of different serological methods to detect antibodies specific to Neospora caninum in bovine and canine sera

Neospora caninum is an apicomplexan parasite responsible for paresis in dogs and abortion in cattle worldwide. Dogs serve as a definitive host, while cattle serve as intermediate host. Many different methods have been developed to detect specific antibodies present in cattle and dog serum. In the present study, the dense granule protein NcGRA6 was incorporated in a latex beads agglutination test (LAT), and compared to other serological methods, including enzyme-linked immunosorbent assay, the direct agglutination test, the immunoblot, and the indirect fluorescent antibody test (IFAT). Using the IFAT as the reference method, 100 sera isolated from Algerian cattle and 100 sera isolated from Algerian dogs, both possibly infected with N. caninum, were used to evaluate the LAT. The sensitivity, specificity, and kappa index were calculated for each host species and assay. For dog sera, the sensitivity and the specificity of the LAT was 76% and 100%, respectively. The McNemar test showed that the LAT was not significantly different from IFAT (P > 0.05). For cattle sera, the sensitivity and the specificity of the LAT were 60% and 100%, respectively. The McNemar test indicated that the LAT was significantly different from IFAT (P < 0.01) and that the LAT was only positive for cattle sera with titers of 1:800 or greater, indicating that LAT can be used for cattle in a clinical context. As well, the LAT has the advantage of being easy and rapid to perform compared to the other assays.

Dual silencing of long and short Amblyomma americanum acidic chitinase forms weakens the tick cement cone stability

This study demonstrates that Amblyomma americanum (Aam) constitutively and ubiquitously expresses the long (L) and short (S) putative acidic chitinases (Ach) that are distinguished by a 210 base pair (bp) deletion in AamAch-S. Full-length AamAch-L and AamAch-S cDNA are 1959 and 1718 bp long, containing 1332 and 1104 bp open reading frames that code for 443 and 367 amino acid residues proteins with the former predicted to be extracellular and the latter intracellular. Both AamAch-L and AamAch-S mRNA are expressed in multiple organs as revealed by qualitative RT-PCR analysis. Furthermore, quantitative reverse transcription polymerase chain reaction analysis revealed that AamAch-L mRNA was downregulated in the mid-gut, but was unchanged in the salivary gland and in other organs in response to feeding. Of significant interest, AamAch-L and/or AamAch-S functions are probably associated with formation and/or maintenance of stability of A. americanum tick cement cone. Dual RNA interference silencing of AamAch-L and/or AamAch-S mRNA caused ticks to loosely attach onto host skin as suggested by bleeding around tick mouthparts and ticks detaching off host skin with a light touch. AamAch-L may apparently encode an inactive chitinase as indicated by Pichia pastoris-expressed recombinant AamAch-L failing to hydrolyse chitinase substrates. Unpublished related work in our laboratory, and published work by others that found AamAch-L in tick saliva, suggest that native AamAch-L is a non-specific immunoglobulin binding tick saliva protein in that rAamAch-L non-specifically bound rabbit, bovine and chicken non-immune sera. We discuss findings in this study with reference to advancing knowledge on tick feeding physiology.

Geo-spatial distribution of serologically detected bovine Foot and Mouth Disease (FMD) serotype outbreaks in Ilesha Baruba, Kwara State-Nigeria

In this study, geo-spatial distribution of bovine Foot and Mouth Disease (FMD) outbreaks were investigated to establish the prevalence and distribution of FMD serotypes in Ilesha Baruba, Kwara state-Nigeria. This purposive study involved 15 FMD outbreaks occurred during December 2010 to March 2011. For geo-spatial analysis, the outbreak locations were indexed using Global Positioning Service (GPS) device (EtrexR). Randomly sampled bovine sera (n=64) from herd representatives were subjected to FMD 3ABC enzyme-linked immunosorbent assay (FMD 3ABC ELISA) and solid-phase competitive ELISA (SP-cELISA), for the screening and serotyping of FMD virus, respectively. The serotypes were indexed with the specific outbreak locations. Through ELISA, the FMD serotypes detected in this study were- serotype O (83%; n=53/64), serotype A (7.8%; n=5/64), serotype vaccine O (1.6%; n=1/64)), and serotype vaccine SAT2 (1.6%; n=1/64). Multiple serotypes were observed in two different combinations; these were O and A (4.7%; n=3/64), and O and SAT2 (1.6%; n=1/64). FMD multiple serotype infections were associated with absence of cross-immunity between serotypes and cross reactivity enhanced by clustered herds, highland study area topography, road and river interconnectivity, possible human settlements, activities and traffic. This study provides baseline information on geo-spatial distribution, and identification of prevalent FMD serotypes in Ilesha Baruba, Kwara state-Nigeria.

Molecular Characterization of a New Babesia bovis Thrombospondin-Related Anonymous Protein (BbTRAP2)

A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite’s growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.

Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle

Mycoplasma bovis causes a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected with M. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity with M. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detected M. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detected M. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.

Significance of a common 65 kDa antigen in the experimental fasciolosis and toxoplasmosis

In the current study, cross-reaction between two important zoonotic parasites; extracellular helminthes Fasciola gigantica and intracellular protozoa Toxoplasma gondii was proved by enzyme linked immunosorbent assay. Five antigens were used to identify and compare the cross-binding activities in the prepared antisera. Two F. gigantica antigens; adult flukes (FgA) and eggs (FgEA) were used to detect IgG in T. gondii naturally infected human sera (TgIHS) and experimentally infected sera of sheep (TgISS), mice (TgIMS) and rats (TgIRS). Three types of T. gondii antigens; RH (TgRHA), local sheep isolate (TgLA) and ME49 isolate (TgMEA) were used to detect cross binding activities in F. gigantica experimental infected rabbit sera (FgIRS) and F. gigantica naturally infected bovine sera (FgIBS). The cross-binding activities in the prepared antisera were strongly directed towards FgA and TgLA rather than the other antigens. The characterization of the five antigens using SDS-PAGE showed 4 common bands of FgA and TgLA; 165, 97, 76, and 65kDa. While two common bands were observed between TgRHA, TgMEA and FgA; 165, and 65kDa. Whereas, two common bands found between three types of T. gondii antigens and FgEA were identified; 165 and 65kDa. The immunogenic cross-reactive bands between FgA and TgLA with F. gigantica infected bovine sera were identified by immunoblot. In FgA, the common immunogenic bands were 165, 65 and 14kDa. While in TgLA, common immunogenic bands were 165 and 65kDa. Whereas, the common immunogenic band between FgA and TgLA identified with T. gondii experimentally infected sheep sera was 65kDa. The current research proves cross reaction between F. gigantica and T. gondii. One common band of 65kDa showed broad immunogenic cross-reactivity with the developed antisera raising the prospect of being putative common immunodiagnostic candidate of both infections.

Trypanosoma vivax GM6 Antigen: A Candidate Antigen for Diagnosis of African Animal Trypanosomosis in Cattle

BackgroundDiagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km2 of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases.Methodology/Principle Findingsthe repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4).Conclusion/Significancethis study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle.

PEROXIDASE-LINKED ASSAY FOR DETECTION OF ANTIBODIES AGAINST BOVINE LEUKOSIS VIRUS

A peroxidase linked assay (PLA) was designed to screen bovine sera for the presence of specific antibodies against bovine leukosis virus (BLV). Out of 201 samples of bovine sera analyzed, 52.2% were considered positive by PLA, 26.4% by AGID, and 38.9% by ELISA. Western blotting analyses excluded 27 samples found to be positive by PLA. PLA showed 100% of sensitivity when compared with AGID and ELISA. Specificity was 64.8% and 78%, respectively (kappa coefficients were 0.70 and 0.83). These findings indicate that PLA can be used as an alternative method for the diagnosis of BLV infection in cattle.

The follow up study after massive outbreak of Akabane and bovine ephemeral fever viruses in Korea

Since a large number of Akabane and bovine ephemeral fever (BEF) infection occurred in the southern part of Korea in 2010, recent information about seroprevalence of Akabane virus (AKAV) and bovine ephemeral fever virus (BEFV) has been required for preventing both diseases. In this study, serological assay against AKAV and BEFV using virus neutralization assay was conducted using 1,743 bovine sera collected from Namwon, Miryang, Yeongju and Uljin which located in Southern part of Korea from March to May in 2012. The overall seropositive rates for AKAV and BEFV were found to be 49.8% and 1.2%, respectively. The regional distribution of seroprevalence for AKAV ranged from 18.1% to 63.7%. Seroprevalences of AKAV were 63.7% in Miryang, 62.3% in Uljin, 50.7% in Namwon, and 18.1% in Yeongju. The seropositive rates for AKAV in southern part of Korea were higher than the annual average at the national level. On the other hand, seropositive rates of BEFV in four regions were from 0.3 to 3.1%. In detail, regional seroprevalences were 3.1% in Miryang, 2.0% in Uljin, and 1.7% in Yeongju, and 0.3% in Namwon. Even only one year after massive outbreaks, overall seropositive rates were very low, similar to the annual average at the nation level. This result indicates that many number of cattle infected with BEFV may be replaced by new born calf or cattle in farm may not be immunized with vaccines. To prevent another epidemic, a national wide warning should be issued and more aggressive control measure must be implied. Recent global warming phenomenon could lead to more vigorous activity of haematophagous vectors and it is possible that arboviral diseases such as AKAV and BEFV are increased. Therefore, continuous sero-monitoring and extensive vaccination combined with control of haematophagous vectors are important to effectively prevent and control diseases caused by AKAV and BEFV.

AC electrokinetics-enhanced capacitive immunosensor for point-of-care serodiagnosis of infectious diseases

The current serological diagnostic method can be time consuming and labor intensive, which is not practical for on-site diagnosis and screening of infectious diseases. Capacitive bioaffinity detection using microelectrodes is considered as a promising label-free method for point-of-care diagnosis, though with challenges in sensitivity and the time “from sample to result.” With recent development in AC electrokinetics (ACEK), especially in dielectrophoresis (DEP), we are able to develop an ACEK enhanced capacitive bioaffinity sensing method to realize simple, fast and sensitive diagnosis from serum samples. The capacitive immunosensor presented here employs elevated AC potentials at a fixed frequency for impedimetric interrogation of the microelectrodes. According to prior work, such an AC signal is capable of inducing dielectrophoresis and other ACEK effects, so as to realize in-situ enrichment of macromolecules at microelectrodes and hence accelerated detection. Experimental study of the ACEK-enhanced capacitive sensing method was conducted, and the results corroborate our hypothesis. The capacitive sensing responses showed clear frequency dependence and voltage-level dependency, which supports the hypothesis that ACEK aids the antigen–antibody binding, and these dependencies were used to optimize our detection protocol. Our capacitive sensing method was shown to work with bovine sera to differentiate disease-positive samples from negative samples within 2min, while conventional immunoassay would require multiple processing steps and take hours to complete. The results showed high accuracy and sensitivity. The detection limit is found to reach 10ng/ml in 2min. The ACEK-enhanced capacitive immunosensor is a platform technology, and can be employed to detect any combination of probe (e.g. antigen) and analyte (e.g. serum antibody) in a small volume of bodily fluids.

Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.

Influence of short-term storage on electrophoretic profile of bovine serum proteins

Storage represents a basic step of the pre-analytical phase for the accuracy of screening tests both in human and veterinary diagnostic laboratories. This study aimed to evaluate the effect of short-term storage on electrophoretic profile of serum proteins in dairy cows. Blood was collected from 18 clinically healthy cows and serum was divided into four aliquots. The first aliquot was immediately analysed (time 0). The second and the third aliquots were stored at +4°C for 24 and 48 h, respectively. The fourth aliquot was frozen at −20° C for 48 hours. A statistical analysis was performed on the obtained data. Albumin and α-globulins relative concentration and A/G ratio showed significant changes after 48 h storage. The results obtained in the current study showed that short-term storage influenced electrophoretic pattern of bovine serum proteins and the A/G ratio. Therefore, the refrigeration of bovine sera for 24 h at the most should be suggested to practitioners in order to obtain reliable serum protein concentrations as useful screening test in veterinary diagnostic laboratory.

Verification of Sera from Cattle and Pigs for Brucellosis with Fluorescence Polarisation Assay

Abstract Fluorescence polarisation assay (FPA) was evaluated as a potential tool improving specificity of serological diagnosis of brucellosis in cattle and pigs. The evaluation was performed by comparing the results of FPA with the results of rose Bengal test (RBT), serum agglutination test (SAT), complement fixation test, and indirect ELISA when problematic sera, regarded as false positive, were tested. Four hundred and seventy-five sera, including 201 porcine and 274 bovine samples, reacting positively in at least one classical serological assay were used. Only six bovine sera were FPA positive (two positive in SAT and RBT and four positive in SAT only). Different situation was observed when porcine sera were examined. Out of 201 sera, 109 were also positive in FPA. The studies confirmed that in cattle FPA enables to reduce highly the number of false positive reactions for brucellosis. On the other hand, in pigs, the method is a little more specific in comparison to other methods applied.

First serosurvey of Besnoitia spp. infection in wild European ruminants in Spain

Besnoitia besnoiti has been reported to affect cattle, wildebeest, kudu and impala, and B. tarandi other wild ruminants (caribou, reindeer, mule deer and musk ox), causing similar characteristic clinical signs and lesions. However, both Besnoitia species have been reported in different geographical areas and the link between the sylvatic and domestic life cycles of Besnoita spp. in wild ruminants and cattle remains unknown. The aim of this study was to evaluate the presence of specific antibodies against Besnoitia spp. in wild ruminants in Spain. A wide panel of sera from red deer (Cervus elaphus) (n=734), roe deer (Capreolus capreolus) (n=124), chamois (Rupicapra pyrenaica) (n=170) and mouflon (Ovis musimon) (n=20) collected from different locations of Spain was analyzed. Beef cattle were present in all sampled areas and, interestingly, bovine besnoitiosis has been widely reported in some of them (e.g., Pyrenees and Central Spain). Sera samples were first examined with an Enzyme-Linked Immunosorbent Assay (ELISA). For red deer and roe deer, the ELISA was standardized with positive and negative control sera from several Cervidae species (100% Se and 98% Sp). Chamois and mouflon sera samples were tested with a previously reported ELISA validated for bovine sera (97% Se and 95% Sp) using protein G as a conjugate. Positive results by ELISA were confirmed a posteriori with a tachyzoite-based Western blot. Sixty-one sera samples from red deer and 17 sera samples from roe-deer were seropositive or doubtful by ELISA. All samples from mouflon were seronegative and 15 sera samples from chamois were considered doubtful. B. besnoiti exposure was only confirmed clearly by Western blot in one red deer and one roe deer from the Spanish Pyrenees where the disease is traditionally endemic.This is the first serological report of Besnoitia spp. infection carried out in European wild ruminants and the results show that specific antibodies are present at least in red deer and roe-deer. Thus, wild ruminants from endemic regions of bovine besnoitiosis should be further studied because they may be putative reservoirs of the parasite.

New flow-through analytical system based on ion-selective field effect transistors with optimised calcium selective photocurable membrane for bovine serum analysis

An analytical system based on ion-selective field effect transistors (ISFETs) with Ca2+ ion selective photocurable membranes is developed to offer a semiautomatic analysis of serum calcium concentration of ruminants. Optimisation of ion-sensitive membrane composition containing different copolymerised plasticizers was performed to minimise the effect of a highly lipophilic samples on sensors characteristics. To improve the system reliability the set of calibration solutions and measurement sequence were also optimised. The system was used to determine total calcium concentration in bovine sera. The precision of the ion determination was higher than reported for double charge ions with a standard deviation of about 3–7%. It is shown that the presence of coagulant in blood serum samples does not affect the determined total calcium concentration.

Potential use of adult bovine serum obtained during the slaughtering process as a biological reagent

Starting from 2013, the Korean ‘Marine Environment Management Act,’ in response to international regulation on marine pollution, will prohibit the marine disposal of land wastes such as animal husbandry by-products and waste produced during the slaughtering process. Currently, most bovine blood is wasted during the slaughtering process and causes environmental problems in Korea. Therefore, long-term measures are required to process blood waste produced during the slaughtering process. The aim of this research is to investigate the potential use of adult bovine serum obtained during the slaughtering process for use in cell cultures. Primary mouse astrocytes, primary human fibroblasts, and L6 cells were cultured with adult male, adult female, and castrated male bovine sera for comparison with fetal bovine serum (FBS). The performance of adult bovine serum was comparable to that of FBS in culture of astrocytes and fibroblasts. Compared to FBS, adult bovine serum supported enhanced proliferation and differentiation of L6 cells. These results suggest that adult bovine serum obtained from the slaughtering process has a potential use as a biological reagent. Moreover, recycling adult bovine serum will contribute to solving the environmental issues arising from the disposal of waste produced during the slaughtering process.

Baculovirus expression and diagnostic utility of the glycoprotein E of bovine herpesvirus-1.1 Egyptian strain “Abu-Hammad”

A recombinant baculovirus construct expressing glycoprotein E (gE) of the Egyptian BoHV-1.1 Abu-Hammad strain (rBac/gE-AbuH) was generated and characterized. The recombinant gE (rgE) secreting protein in culture medium of infected insect cells was used as a coating antigen in an indirect enzyme-linked immunosorbent assay (ELISA) to test its utility for detection of antibody against gE of BoHV-1. Indirect gE-ELISA was compared to standard virus neutralization test and commercial blocking gE-ELISA for detection of anti-gE antibody in a panel of bovine sera. Antibody titers estimated by both ELISAs were closely correlated with those determined by virus neutralization test. In conclusion, the developed indirect gE-ELISA was a reliable candidate for inexpensive detection of anti-gE antibody in control and experimental bovine sera with high specificity and sensitivity. Moreover, it emphasized the diagnostic utility of gE based ELISAs to distinguish cattle infected with BoHV-1 from those vaccinated with the gE negative mutants.

Validation of a Commercially Available Indirect Elisa Using a Nucleocapside Recombinant Protein for Detection of Schmallenberg Virus Antibodies

A newly developed Enzym Like Immuno Sorbant Assay (ELISA) based on the recombinant nucleocapsid protein (N) of Schmallenberg virus (SBV) was evaluated and validated for the detection of SBV-specific IgG antibodies in ruminant sera by three European Reference Laboratories. Validation data sets derived from sheep, goat and bovine sera collected in France and Germany (n = 1515) in 2011 and 2012 were categorized according to the results of a virus neutralization test (VNT) or an indirect immuno-flurorescence assay (IFA). The specificity was evaluated with 1364 sera from sheep, goat and bovine collected in France and Belgium before 2009. Overall agreement between VNT and ELISA was 98.9% and 98.3% between VNT and IFA, indicating a very good concordance between the different techniques. Although cross-reactions with other Orthobunyavirus from the Simbu serogroup viruses might occur, it is a highly sensitive, specific and robust ELISA-test validated to detect anti-SBV antibodies. This test can be applied for SBV sero-diagnostics and disease-surveillance studies in ruminant species in Europe.