Sheep Serum Research Articles

Cross-sectional sero-epidemiological survey of sarcoptic mange in sheep of Western Castile and Leon, Spain

The aim of the present study was to determine the prevalence of Sarcoptes scabiei-infection of ovine livestock in three provinces (Leon, Zamora and Salamanca) in the Western part of the Castile and Leon region in Spain, and to determine the association between different variables and seropositivity. A total of 3730 sheep sera from 373 flocks (10 sera from each flock) collected from May to September over the course of the years 2006 and 2007 were individually analysed by an indirect antibody ELISA validated for diagnosing sarcoptic mange in sheep. The overall flock-level true prevalence was 22.6% (95% CI: 17.8–27.4), the overall individual-level true prevalence within the total flocks was 7.2% (95% CI: 6.1–8.3) and the overall individual-level true prevalence within the seropositive flocks was 31.3% (95% CI: 27.2–35.4). The apparent prevalences, at flock-level and at individual-level within the total flocks and within the seropositive flocks, were not statistically different ( p > 0.05) when the primary production objective of the flock is milk vs. meat, or in smaller (≤276 sheep, 50th percentile) vs. larger flocks (>276 sheep). The apparent prevalences, at flock-level and at individual-level within the seropositive flocks, were, likewise, not statistically different between the three provinces, but the individual-level apparent prevalence within the total flocks showed significant variation from one province to another ( p ≤ 0.05). Sheep maintained in the Provinces of Zamora and Salamanca had greater odds (OR = 1.7, 95% CI: 1.2–2.6; OR = 1.9, 95% CI: 1.3–2.8, respectively) of being seropositive than those located in Leon Province (OR = 1.0). The findings of the present study clearly show the need to implement in this region effective control measures against sarcoptic mange in sheep.

Serological, Bacteriological, and Molecular Diagnosis of Brucellosis in Domestic Animals in Croatia

To present the surveillance data on Brucella melitensis, B. suis, and B. ovis infection in cattle, sheep, goats, and swine in Croatia obtained in 2008 by serological, bacteriological, and molecular methods for diagnostics of brucellosis in domestic animals. We serologically tested 42,785 cattle serums, 22,686 sheep and goat serums, and 28520 swine serums using the Rose Bengal test, complement fixation test, and various immunosorbent assays. We also tested 10,173 ram blood samples for B. ovis infection using the complement fixation test. Bacteriological examination was conducted on 214 samples collected from 34 serologically positive animals. Different molecular methods were employed in the identification and typing of 20 isolates from the samples. B. melitensis biovar (bv.) 3 was confirmed with different identification methods in 2 flocks in 2 Croatian counties and B. suis bv. 2 in 3 flocks in 3 counties. B. melitensis in cows was confirmed for the first time in Croatia. Infection with B. ovis was serologically confirmed in 202 rams in 12 counties. In 2008, the size of the brucellosis-affected area in Croatia and the efficiency of detection and prevention of brucellosis in sheep, goats, and swine were satisfactory. Infection with B. melitensis in cattle was confirmed for the first time and possible links for infection in humans were detected. More efficient measures for suppression and control of ovine epididymitis are required and a new strategy may be necessary for complete eradication of this disease.

Potency against enterotoxemia of a recombinant Clostridium perfringens type D epsilon toxoid in ruminants

Enterotoxemia, a disease that affects domestic ruminants, is caused mainly by the epsilon toxin from Clostridium perfringens type D. Its eradication is virtually impossible, control and prophylaxis are based on systematic vaccination of herds with epsilon toxoids that are efficient in inducing protective antibody production. The use of recombinant toxins is one of the most promising of these strategies. This work evaluates the potency of a Cl. perfringens type D epsilon toxoid expressed by Escherichia coli administered to goats, sheep, and cattle. The etx gene was cloned into the pET-11a plasmid of E. coli strain BL21 to produce the recombinant toxin. Rabbits ( n = 8), goats, sheep, and cattle ( n = 5 for each species) were immunized with 0.2 mg of the insoluble recombinant protein fraction to evaluate vaccine potency of the epsilon toxoid studied. Antibody titers were 40, 14.3, 26, and 13.1 IU/mL in the rabbit, goat, sheep, and cattle serum pools, respectively. The epsilon toxoid produced and tested in this work is adequate for immunization of ruminants against enterotoxemia.

Anti-allergic, anti-pruritic, and anti-inflammatory activities of Centella asiatica extracts.

This study investigated antipruritic and anti-inflammatory effect of Centella asiatica extract in rats and anti-allergic in vitro using sheep (Capra hircus) serum method and compound 48/80 induced mast cell degranulation method, compared with standard drug ketotifen fumarate. In rats, extract of Centella asiatica administered orally was examined for anti-pruritic study and chlorpheniramine maleate was used as standard drug while carageenan paw induced inflammatory method was used for the antiinfammatory study. The results show that the extracts of Centella asiatica exhibited antiallergic, anti-pruritic and anti-inflammatory activities.

PP-198 Comparison between different immunological techniques for detection of circulating Fasciola antigen in sheep

The detection of Fasciola antigen in serum or stool could be more valuable in diagnosis, hence early treatment before irreparable damage. In this study, fresh adult Fasciola gigantica worms were collected, then incubation in culture medium and collected medium was used to extract crude excretory-secretory (E/S) antigen. E/S was used to immunize rabbits to raise specific antibodies against Fasciola spp. Purified antibodies are further used as primary capture to coat ELISA plates. The secondary capture of antibodies was by conjugation with horse reddish peroxidase. Sandwich ELISA and DOT-ELISA were performed to detect Fasciola antigens in both serum and stool samples collected from a total of 152 sheep. After slaughtering, gross inspection of liver and parasitological stool examination, sheep were divided into Fasciola positive group (97 sheep), other helminthic infection group (30 sheep) and healthy control group (30 sheep). Fasciola antigen detected in serum of sheep by ELISA showed 94.8 % sensitivity and 95 % specificity. Copro-antigen detected by ELISA showed 96.9% sensitivity and 96.7% specificity. The sensitivity and specificity of coproantigen by ELISA in stool sample were higher than that recorded by Sandwich ELISA for serum. Dot ELISA sensitivity was found to be 98.9% and specificity 98.3%. In conclusion the Dot ELISA gives better sensitivity and specificity than sandwich ELISA for serum and coproantigen in stool by ELISA. (New York Science Journal 2010;3(7):34-39). (ISSN: 1554-0200).

Electrophoretically mediated microanalysis for the evaluation of interspecies variation in cholinesterase metabolism

This study describes an electrophoretically mediated microanalysis method, suitable for the preclinical evaluation of the hydrolysis of ester drugs by the serum of different animals and for further characterization of human-animal correlation. Dog, cat, cow, horse, sheep, rat and human serum were diluted (25%) in the appropriate buffer and replaced the enzyme solution usually used in electrophoretically mediated microanalysis methods for the study of enzyme kinetics. They were then compared in terms of the ability to hydrolyze acetylthiocholine and butyrylthiocholine (0.25 mM) by in-capillary reaction. Human serum afforded the highest conversion rates (52% butyryltiocholine and 34% acetylthiocholine) followed by horse (31 and 35%), dog (26 and 24%), cat (22 and 14%), rat (11 and 15%) and sheep (8 and 8%). Hydrolysis by bovine serum was negligible. The method is fast (under 8 min including rinsing steps), sensitive (under 25 microM substrate could be quantified) and repeatable (RSD approximately 2%), only requiring minute amounts of sample.

Effects of proBDNF on cell proliferation and differentiation in hippocampal dentate gyrus in Alzheimer' disease rat model

To explore the effects of proBDNF on cell proliferation and differentiation in hippocampal dentate gyrus in Alzheimer' disease (AD) rat model. The AD rat model was established. Alzet osmotic minipumps were connected to right hippocampus of AD rat and filled with proBDNF, sheep antibody to proBDNF or normal sheep serum respectively. Rats received the injection for 14 days at the speed of 0.5 microl/h. 5-bromo-2'-deoxyuridine (BrdU, 50 mg/kg, ip) was injected twice daily for 14 days. BrdU immunohistochemistry was processed to determine the number of newly generated cells. To examine the phenotype of newly generated cells, immunofluorescent triple labeling was conducted to colocalize BrdU-positive cells with rabbit anti-doublecortin (DCX) or mouse anti-glial fibrillary acid protein (GFAP). proBDNF group had fewer BrdU positive cells in dentate gyrus (P < 0.01), while anti-proBDNF group had more BrdU positive cells (P < 0.01) as compared with control group respectively. Immunofluorescent triple labeling showed that there was no phenotypic difference of BrdU positive cells between each group. proBDNF can suppress the proliferation of hippocampal neuron in dentate gyrus in AD rats while anti-proBDNF has the opposite effect. These findings suggest that promoting the hippocampal neurogenesis by blocking the functions of endogenous proBDNF may be a potential therapeutic strategy for AD.

Identification of potential vectors of and detection of antibodies against Rift Valley fever virus in livestock during interepizootic periods

To evaluate the prevalence of Rift Valley fever virus (RVFV) antibodies in livestock and presence of competent mosquito vectors of RVFV during an interepizootic period (IEP) in Kenya. 208 sheep and 84 goats ranging in age from 4 months to 15 years, from 2 breeding herds. Blood specimens were collected from the sheep and goats during the 1999-2006 IEP in Rift Valley Province, and serum was harvested. Serum specimens were tested for IgG and IgM antibodies against RVFV by use of an ELISA. In addition, 7,134 mosquitoes were trapped in Naivasha, Nairobi, and Northeastern Province, and speciation was performed. No animals were seropositive for IgM against RVFV. Of the animals born after the 1997-1998 epizootic, 18% (34/188) of sheep were seropositive for IgG against RVFV, compared with 3% (2/75) of goats. Seventy percent (8,144/11,678) of the mosquitoes collected were of the Culex subgenera; 18% (2,102/11,678) were Aedes spp. Detection of IgG in the sera of sheep and goats born after the 1997-1998 epizootic and before the 2006 epizootic indicated that virus activity existed during the IEP. Detection of Aedes mosquitoes, which are competent vectors of RVFV, suggested that a cryptic vector-to-vertebrate cycle may exist during IEPs.

Toxoplasma gondii in wild cervids and sheep in Finland: North-south gradient in seroprevalence

A nationwide seroepidemiological study was conducted to estimate the prevalence of Toxoplasma gondii in selected wild and domestic ruminants in Finland. Serum samples from 1367 game cervids collected during the hunting season in 2008-2009 and 1940 sheep sera collected in 2008 were screened with a commercial direct agglutination test at a serum dilution of 1:40. T. gondii-specific IgG antibodies were detected in 116 (9.6%) of 1215 moose (European elk, Alces alces), 36 (26.7%) of 135 white-tailed deer (Odocoileus virginianus), 3 (17.6%) of 17 roe deer (Capreolus capreolus), and 477 (24.6%) of 1940 domestic sheep. Seropositive sheep were found in 74 (76.3%) of the 97 flocks examined. The odds of seropositivity in the adult moose was 2.9 times higher than the odds in calves; in white-tailed deer, the odds ratio was 3.2. The male moose had a significantly lower seroprevalence than the female, whereas the seroprevalence in the male white-tailed deer was higher than in the female; the odds ratios were 0.6 and 2.5, respectively. A clear geographical gradient in the seroprevalence was revealed in moose and sheep. The seroprevalences were lowest (1.6 and 8.6%, respectively) in the north and highest (24.6 and 36.4%, respectively) in the south-western regions, and ranged between these values in the other regions. In fact, the seroprevalence in moose from the south-west was not significantly different from the prevalence in white-tailed deer from the same area. Thus, the Finnish wild cervids and sheep are commonly exposed to T. gondii, especially in the southern part of the country.

Measurement of monoterpenes and sesquiterpenes in serum, plasma, and rumen fluid from sheep

Studies involving the consumption, metabolism, and elimination of terpenes by small ruminants consuming terpene-laden shrubs as well as those exploring the potential for natural products as rumen modifiers could benefit from a procedure that measures terpenes in both blood and rumen fluid and that is suitable for either serum or plasma. The objective of this study was to modify an existing procedure for plasma utilizing solid phase extraction/gas chromatography, and extend its use for measurement of structurally diverse mono- and sesquiterpenes in three fluids (serum, plasma, and rumen fluid) from sheep. Generally, terpene recovery was lower from rumen fluid than from serum or plasma, although the extent and direction of differences varied among chemicals. Fourteen terpenes (camphene, β-pinene, α-terpinene, p-cymene, cis-β-ocimene, 1,8-cineole, γ-terpinene, terpinolene, linalool, camphor, longifolene, β-caryophyllene, α-humulene, and caryophyllene oxide) were recovered from serum at near unity. Recovery from rumen fluid was lower than that for serum or plasma for most terpenes, but eight ( p-cymene, 1,8-cineole, cis-sabinene hydrate, terpinolene, borneol, terpin-4-ol, α-terpineol, and caryophyllene oxide) were recovered at near unity. Yet, 15 terpene recoveries were below 0.75 ng/ng (tricyclene, α-pinene, camphene, sabinene, β-pinene, myrcene, 2-carene, 3-carene, α-terpinene, cis-β-ocimene, limonene, γ-terpinene, longifolene, β-caryophyllene, and α-humulene). Oxygenated monoterpenes were typically recovered in greater quantities and hydrocarbon monoterpenes were least effectively recovered with this method. This procedure allows for simultaneous measurement and recovery adjustment of a number of terpenes from serum, plasma, and rumen fluid of sheep.

Immunoaffinity chromatographic analysis for purification of specific diagnostic antigens of Paramphistomum epiclitum

Polypeptide profile of somatic antigen of Paramphistomum epiclitum (PSAg) and Gastrothylax crumenifer (GSAg) was studied by SDS-PAGE. PSAg and GSAg showed 14 and 19 polypeptides in the range of 14.9-95.5 and 13.7-129.6kDa with six common polypeptides of mol wt 16.8, 21.8, 23.7, 35.5, 43.4 and 70.8kDa. P. epiclitum experimentally infected sheep sera were used for identification of specific immuno-dominant peptide in the range of 37-40kDa against P. epiclitum by western blotting. Hyperimmune sera (HIS) was raised in rabbit against the identified polypeptide, IgG was separated from HIS and an immunoaffinity column was constructed with a binding percentage of 83.74 of IgG with CNBr activated Sepharose 4B. Purification of somatic antigen (PSAg) was done with immunoaffinity chromatography and 37-40kDa protein antigen was isolated in pure form with recovery percentage of 2.97%. This purified fraction of somatic antigen can be used as a candidate antigen for development of serological assay for early diagnosis of paramphistomosis among livestock.

Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (Npro) protein of bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (N(pro)) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV N(pro)-His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the N(pro) protein was confirmed by western blot using anti-BVDV hyperimmune cattle, sheep and goat serum, and anti-N(pro) rabbit serum. When rabbits were immunized with the N(pro) protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite N(pro)-specific antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the field cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for N(pro) antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV N(pro) protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.

Cross-Sectional, Longitudinal and Prospective Epidemiological Studies of Rift Valley Fever in Al-Hasa Oasis, Saudi Arabia

Rift Valley Fever (RVF) is a disease affecting ruminants and humans in many countries of the African continent, and was reported for the first time in 2000 in the south-west region of Saudi Arabia, and north-west region of Yemen. In Saudi Arabia, RVF was not reported outside the south-west region. However, its situation outside the affected region is not properly and scientifically clarified. In this investigation cross-sectional and longitudinal serological studies were conducted on domestic ruminants in Al-Hasa Oasis east of Saudi Arabia, to clarify the freedom or presence, prevalence, and distribution of Rift Valley fever (RVF). A total of 598 serum samples were collected from sheep (225), goats (102), cattle (242), and camels (29) during the year 2007. Enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of anti-RVF IgG and IgM antibodies in sheep, goats, and cattle sera. Camels sera were tested for the presence of anti-RVFV antibody by isotype-nonspecific inhibition ELISA (iELISA). Two out of 598 (0.3%) sera were IgG, but not IgM, seropositive, other samples were IgG and IgM seronegative. Both seropositives were ewes, more than three years old, and located about 45 kilometers apart. For the longitudinal study a single sentinel herd of 40 sheep were established in the Oasis. These animals were subjected to daily clinical observation and monthly blood sampling. This investigation lasted 35 month starting August 2005. Sera from the blood samples were tested for the presence of IgG using the ELISA technique. There were no clinical signs observed on the animals that may suggest RVF. Sera testing also failed to detect IgG against RVF virus.

Leptospira wolffii, a potential new pathogenic Leptospira species detected in human, sheep and dog

Leptospirosis is the most common zoonotic disease, which is transmitted to humans through contaminated water or direct exposure to the urine of infected animals. In this study, the presence and prevalence of Leptospira species in the infected samples of human (n=369) and sheep (n=75) sera and also dogs' urine (n=150), collected from four provinces of Iran, were investigated by using nested-PCR/RFLP assay followed by sequencing analysis. Nested-PCR assay detected that 98/369 (26.5%) human, 13/75 (17.33%) of sheep's sera and 33/150 (22%) dogs' urine samples were positive for Leptospira DNA. RFLP assay detected that all positive cases had either pathogenic or intermediate Leptospira species. By sequence analysis, Leptospira interrogans was the most prevalent species among the examined samples of human (53/82, 64.6%) and sheep (11/13, 84.6%). However, in dog samples, Leptospira wolffii (27/29, 93.1%) was detected for the first time and was the dominant species. The presence of L. wolffii with 100% identity in clinical human samples and animals suspected with Leptospira may provide evidence for circulation of L. wolffii and its role in transmission cycle within human and animal hosts. In addition, this species can be potentially pathogenic to human and probably animal hosts. A large epidemiology survey would be needed to define the presence and the prevalence of this species in global endemic regions.

Antibacterial Activity of Some Plant Extracts on Escherichia coli with Special Reference to its Resistance Pattern

The present work was planned to isolate, characterize and evaluate the prevalence of E. coli serotypes in 180 samples (herbs, water and chicken) as well as detection of haemolysin production, Congo Red binding activity, serum activity and antimicrobial susceptibility test. Forty four samples out of 180 samples were found to be positive to E. coli with a percentage of 24.4%. The serological identification showed that O125, O112 and O86 were the most prevalent serotype among the 44 isolated strains with percentages of 27%, 25% and 18% respectively. The study showed that all 44 E-coli isolates had the ability to bind with C.R. dye gave red colonies and were sensitive to bactericidal effect of human and sheep serum (100%). The results of haemolysis showed that the isolates belonged to O112 and 0114 serotypes had a hemolytic activity and the other serotypes had no hemolytic activity. The result of antibiotics sensitivity indicated that E. coli strains were highly resistant to penicillin and erythromycin (100%), followed by gentamicin, ampicillin, oxytetracycline and nalidixic acid with percentages of 81.8%, 75%, 47.7% and 15.9 %respectively. All E. coli strains were found to be moderately sensitive to ceftazidime (100%) and sensitive to ciprofloxacin (70.5%). The result of ethanolic plant extract showed that Coriander, Ginger, bay leaf, Black pepper, Chilly, Parsley and Turmeric did not show any bactericidal activity against E. coli strains. While Hibiscus, Thyme and Cinnamon showed intermediate bactericidal activity in concentration 100%. Moreover, Black cumin showed low bactericidal activity against herbs and water isolates only with 100% concentration.

Recombinant OMP28 antigen-based indirect ELISA for serodiagnosis of bovine brucellosis

Brucellosis is a zoonosis of both public health and economic importance in many developing countries including India. Early detection and segregation of the infected animals are important in order to control the disease. Serodiagnostic tests for brucellosis is mainly based on detection of antibodies developed against lipopolysaccharide (LPS) component of cell. In this study we evaluated a protein antigen, 28 kDa outer membrane protein (OMP28), of Brucella melitensis as an alternative to LPS. Recombinant OMP28 was produced in Escherichia coli system. The efficacy of purified OMP28 was studied in an indirect enzyme-linked immunosorbent assay (ELISA) for diagnosis of brucellosis in field sera collected from different regions of country. Using known negative and known positive serum samples it was found that OMP28 is immunoreactive to Brucella infected cattle, sheep, goat and dog sera. Three hundred and eighty two cattle sera were screened by OMP28 antigen-based ELISA and the results were compared to rose Bengal plate agglutination Test (RBPT). Recombinant OMP28 antigen-based ELISA has shown sensitivity of 88.7%, specificity of 93.8% and accuracy of 92.9%. It was concluded that recombinant B. melitensis OMP28 could be used as a protein antigen for diagnosis of brucellosis in domestic animals.

334 EFFECT OF A GROWTH MEDIUM DURING IVM ON EMBRYO DEVELOPMENT OF PREPUBERTAL EWE OOCYTES

The aim of this study was to assess the effect of an in vitro growth medium (De Wu et al. 2006 Biol. Rep. 75, 547-554) in prepubertal ewe oocytes selected by the brilliant cresyl blue (BCB) test. Prepubertal ewe oocytes were recovered by slicing ovaries of slaughtered animals and immediately exposed during 1 h to 13 μM BCB and classified according to their cytoplasm coloration (Rodriguez-Gonzalez E et al. 2002 Theri- ogenology 57(5), 1397-1409): BCB+ (blue cytoplasm, hypothetically grown oocytes) and BCB- (uncolored cytoplasm, hypothetically growing oocytes). Uncolored oocytes (BCB-) were matured using three culture media: growth medium (GM: TCM-199, 0.04 μg mL-1 FSH, 0.04 μg mL-1 LH, 0.004 μg mL-1 estradiol, 100 μg mL-1 ascorbic acid, and 5 μL mL-1 ITS: insulin transferrin selenium), conventional maturation medium (CM: TCM-199, 10 μg mL-1 FSH, 10 μg mL-1 LH and 1 μg mL-1 estradiol) and modified maturation medium (MM: CM with the addition of 100 μg mL-1 ascorbic acid and 5 μL mL-1 ITS). Oocytes were matured in GM for 12 h and then separated into 2 groups, CM (GM+CM) and MM (GM+MM) for another 12 h of maturation. Two extra groups of BCB- oocytes were directly cultured for 24 h in CM or MM media (BCB-/CM and BCB-/MM). Colored oocytes (BCB+) and a control group (oocytes not exposed to BCB) were matured for 24 h in CM. All groups were cultured at 38.5°C and 5% CO2 in humidified atmosphere. Fertilization took place in SOF medium supplemented with 10% of estrous sheep serum during 20 h with a sperm concentration of 1 × 106 spermatozoa/mL. Presumptive zygotes were cultured for 8 days in SOF with 10% FCS at 38.5°C, 5% CO2 and 5% O2. Results are shown in Table 1. The percentage of morula plus blastocyst obtained from BCB - oocytes was significantly increased in oocytes exposed to growth medium (containing ITS, ascorbic acid and low hormone concentrations; groups GM+CM and GM+MM) for the first 12 h. An increasing tendency has also been observed in blastocyst yield in these two groups. Regarding maturation rate, no differences were found in all groups (data not shown). In conclusion, as De Wu et al. (2006) showed in prepubertal gilts, we also achieved some improvements in embryo development of growing oocytes when the first 12 h of maturation took place in a growth medium. However, embryo developmental potential of BCB- oocytes is still lower compared with that of BCB+ oocytes. Table 1.Effect of GM on embryo development of BCB- oocytes Grant sponsor Spanish Ministry of Science and Innovation.Code: AGL2007-60227-CO2-01

Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRalpha1 ECD, we generated a model of the human GlyRalpha1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRalpha1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.

Standardization and application of indirect plate ELISA for immunodiagnosis of paramphistomosis in ruminants

Indirect plate enzyme-linked immunosorbent assay was standardized and evaluated for its effectiveness in immunodiagnosis of paramphistomosis in experimental and clinical cases in sheep, goat, cattle and buffaloes by using somatic whole adult antigen of Paramphistomum epiclitum and Gastrothylax crumenifer. Plate enzymelinked immunosorbent assay (ELISA) was standardized using 2 μg/ml of antigen concentration with 1:200 and 1:1,000 of sera and conjugate dilution. Indirect Plate ELISA was able to demonstrate the antibody titre at different weeks postinfection in experimental sheep. Immune response at weekly interval varied in all the four experimental sheep. A paired t-test between two types of somatic antigens (P. epiclitum and G. crumenifer) showed that experimental sheep sera showed more affinity for homologous antigen as compared to heterologous antigen. A comparison of plate ELISA on suspected field sera and fecal samples examination by sedimentation method revealed that 77 samples were found to be positive by ELISA but only seven by fecal examination. Sensitivity of plate ELISA was found to be 85.71%, whereas specificity was 23.65% indicating that this test is quite sensitive for clinical cases; an early diagnosis, however, lacks specificity. In comparison to ELISA test the sensitivity and specificity of fecal examination were 7.79 and 88%, respectively.

LINFADENITE CASEOSA EM REBANHOS OVINOS NO ESTADO DE MINAS GERAIS, BRASIL: PREVALÊNCIA E INFORMAÇÕES DE MANEJO

Corynebacterium pseudotuberculosis (Cp) is the etiological agent of caseous lymphadenitis (CL) and an economically important disease that affects sheep. Seroprevalence of pseudotuberculosis and risk factors associated with CL were studied in sheep herds from Minas Gerais, Brazil. Blood samples were collected from 642 sheep from 97 flocks and sera was tested for antibodies by ELISA. A questionnaire was applied in order to gather information about the farm, flock, and animals, such as breed, sex and age. This is the first seroepidemiological survey for CL in sheep flocks from Minas Gerais. Estimated animal prevalence was 70,9% (95% CI: 64,7 - 77,0%) and prevalence of infected flocks was 95,9% (95% CI: 89,8 - 98,9%). These values were considerably higher than those predicted in the questionnaire. Seropositivity frequency in sheep sera varied significantly depending on breed (P=0,021), and age (P=0,049), and the highest rates were observed in animals older than 12 months.