High OD Values Research Articles

Serum Samples from Co-Infected and Domestic Cat Field Isolates Nonspecifically Bind FIV and Other Antigens in Enzyme-Linked Immunosorbent Assays.

We evaluated enzyme-linked immunosorbent assay (ELISA) specificity for measuring seroantibody responses to two types of retroviral infections in domestic cats: feline immunodeficiency virus (FIV) and feline foamy virus (FFV). We compared the seroreactivity of specific pathogen-free (SPF) cat sera, sera from SPF cats inoculated with either FIV or FFV, and field isolates (e.g., shelter or privately owned cats). Sera from SPF cats experimentally infected with the cognate virus had significantly lower background in both FIV and FFV ELISAs compared to sera from negative field isolates. ELISA values for SPF cats exposed to either FIV or FFV tended to have higher OD values on the opposite ELISA antigen plate. FIV nonspecific background absorbance was greater than that of FFV, and 10 of 15 sera samples from FIV seronegative field samples were measured in the indeterminant range. These findings highlight that exposure to off-target pathogens elicit antibodies that may nonspecifically bind to antigens used in binding assays; therefore, validation using sera from SPF animals exposed during controlled infection results in the setting of a cutoff value that may be inappropriately low when applied to field samples. Our work also suggests that infection of domestic cats with pathogens other than FIV results in antibodies that cross-react with the FIV Gag antigen.

Evolutionary Mechanism of Immunological Cross-Reactivity Between Different GII.17 Variants.

Human norovirus is regarded as the leading cause of epidemic acute gastroenteritis with GII.4 being the predominant genotype during the past decades. In the winter of 2014/2015, the GII.17 Kawasaki 2014 emerged as the predominant genotype, surpassing GII.4 in several East Asian countries. Hence, the influence of host immunity response on the continuous evolution of different GII.17 variants needs to be studied in depth. Here, we relate the inferences of evolutionary mechanisms of different GII.17 variants with the investigation of cross-reactivity and cross-protection of their respective antisera using the expression of norovirus P particles in Escherichia coli. The cross-reactivity assay showed that the antisera of previous strains (GII.17 A and GII.17 B) reacted with recent variants (GII.17 C and GII.17 D) at high OD values from 0.8 to 1.16, while recent variant antisera cross-reacting with previous strains were weak with OD values between 0.26 and 0.56. The cross-protection assay indicated that the antisera of previous strains had no inhibitory effect on recent variants. Finally, mutations at amino acids 353–363, 373–384, 394–404, and 444–454 had the greatest impact on cross-reactivity. These data indicate that the recent pandemic variants GII.17 C and GII.17 D avoided the herd immunity effect of previous GII.17 A and GII.17 B strains through antigenic variation.

SUN-LB5 GnRHR ECL-2 Epitopes Targeted by Activating Autoantibodies in Polycystic Ovary Syndrome

Background: Activating autoantibodies (AAb) are directed to the gonadotropin releasing hormone receptor second extracellular loop (GnRHR-ECL2) and are pathogenic when induced in rats. We previously reported GnRHR-ECL2-AAb were elevated in sera from patients with PCOS (Rotterdam criteria) compared to ovulatory infertile controls (OIC). Methods:Human studies: ELISA detection of GnRHR Abs used a synthetic h-GnRHR-ECL2 28 mer peptide (LifeTein) as the target antigen. We assayed AAb activity in GnRHR transfected cells using a GeneBLAzer FRET assay (Invitrogen). ELISA AAb epitope locations on the ECL2 were identified on a minipin plate (pins 1-11 containing sequential 2 aa offsetting octapeptides, Mimotope, Inc) using sera from 30 PCOS subjects, 33 OIC and 18 normal controls (NC). Results:Human sera: An ELISA assay for GnRHR-ECL2-AAbs in the PCOS group was markedly higher than the NC group (P<0.0001) and the OIC subjects (P<0.003). The minipin data demonstrated one or more positive OD peaks on pins 4 (20%), 5 (47%) and 8 (47%) which shared L-aa sequences FSQC or CSFSQ. OIC had only 5 subjects with peaks at minipins 4 or 5 and NC had only 3 lower peaks and one with higher OD values over all minipins. GnRHR-AAb Specific Activity (SA) was estimated by measuring serum activity before and after suppression of AAb sensitive activity by addition of retro inverso D-aa (RID) peptides. These were specifically designed to mimic and decoy the AAb L-aa epitope sequences of pins 5 and 8). SA was measured in 10 selected PCOS and 10 OIC subjects who had positive ELISA values. The baseline activity in the PCOS group was significantly higher than OIC (P < 0.01) and dropped 50% with preincubation with peak 5 RID and 25% with peak 8 RID. The addition of both peak 5 and peak 8 RID suppressed the PCOS group activity to OIC levels (P > 0.2). There was no significant change in activity in the OIC subjects by the addition of peaks 5 + 8 RID peptides. Conclusion: These PCOS GnRHR-AAb data confirm the presence of significant activation of the GnRHR by AAb targeted to specific epitope(s) proximate to the disulfide GnRHR-ECL2 linkage to the nearby ECL1. These data are compatible with a pathophysiological role for GnRHR-AAb in PCOS and may provide both diagnostic and therapeutic opportunities.

Recombinant vaccine protects juvenile hybrid grouper, Epinephelus fuscoguttatus × Epinephelus lanceolatus, against infection by Vibrio alginolyticus

Vibrio alginolyticus has been recognized as one of the most significant bacterial diseases in grouper species. In this study, two outer membrane proteins of V. alginolyticus, the OmpK and OmpW, were considered for vaccine development, since both OmpK and OmpW were proven by previous studies to be promising vaccine candidates. Recombinant vaccines were constructed by cloning the OmpK and OmpW genes of V. alginolyticus into pET32 Ek/LIC vector and expressed in Escherichia coli. Fish were immunized with inactivated E. coli expressing the OMPs. Juvenile hybrid groupers, Epinephelus fuscoguttatus × Epinephelus lanceolatus, were divided into five groups of 150 fish per group. Group 1 was vaccinated IP with rOmpK while group 2 with rOmpW; group 3 was vaccinated with host E. coli BL21(DE3), group 4 with phosphate-buffered saline (PBS) only, and group 5 was the environmental control group. Each vaccination was carried out with 0.5 mL fish−1 vaccine and at 28 °C water temperature. Booster dose of respective vaccine was delivered on day 14, and all groups were challenged with 109 CFU mL−1 of live V. alginolyticus using intraperitoneal (IP) injection at 0.5 mL fish−1 on day 28. The relative percentage survival (RPS) was highest for rOmpK group at 100%, followed by rOmpW group at 63%, and E. coli BL21(DE3) group at 0%. The IgM antibody specific to V. alginolyticus was detected in the serum, in which both rOmpK and rOmpW vaccinated groups showed an increasing pattern in antibody level following vaccinations. The IgM against rOmpK showed significantly (p < 0.05) higher OD values than rOmpW at challenge on day 28 post vaccination. The results of this study suggest that rOmpK succeeded in providing excellent protection while the rOmpW succeeded in providing partial protection against infection by V. alginolyticus.

A Novel PF4-Dependent Platelet Activation Assay Identifies Patients Likely to Have Heparin-Induced Thrombocytopenia/Thrombosis (HIT)

Antibodies (abs) specific for heparin/platelet factor 4 (H/PF4) complexes are a hallmark of heparin-induced thrombocytopenia and thrombosis (HIT) and are thought to play a role in predisposing patients to thrombocytopenia and thrombosis. A solid phase assay that uses H/PF4 complexes as targets for ab detection (PF4 ELISA) is almost invariably positive in patients with HIT. This test can be performed quickly and a negative result makes HIT very unlikely; however, many patients testing positive in this assay do not develop HIT. A second test, the serotonin release assay (SRA), considered the "gold standard" for HIT diagnosis, selectively detects HIT abs that activate platelets and are presumptively pathogenic. A positive SRA test correlates well with clinical HIT but the SRA is technically demanding and is routinely performed only by a few reference laboratories, precluding its use for early patient diagnosis and management. This can have adverse consequences since both over- and under-diagnosis of HIT can have serious medical and economic impact.We recently showed that platelet-activating HIT abs preferentially recognize PF4 bound to platelet glycosaminoglycans in the absence of heparin (Blood 2015; 125(1): 155-61). Based on this finding we developed a technically simple assay (PF4-dependent p-selectin expression assay [PEA]) for detection of platelet-activating HIT abs. In the PEA, target platelets are first "primed" for activation by being incubated with PF4 and are then exposed to patient serum. P-selectin expression (percent of maximum) is measured as an indicator of platelet activation (Thromb Haemost. 2015; 114(5) epub). Specificity of a positive test result is confirmed by inhibition with high dose heparin (HDH).To compare diagnostic performance of the PEA and the SRA, 91 sera from patients referred for HIT testing who had been assigned clinical scores for HIT ("4T scores") ranging from 0 to 8 (low to high likelihood of HIT, respectively) were tested in both assays. Samples from patients with Intermediate or High 4T scores (4-8) and PF4 ELISA OD values >1.0 were considered "HIT-positive " given that neither criterion by itself has a high positive predictive value for HIT. Intermediate to High 4T-scored patients with PF4 ELISA ODs <1.0 were considered "HIT-negative ", as were samples associated with low 4T scores, which are known to have a high negative predictive value for HIT.Receiver operating curve (ROC) analysis showed that the PEA had greater accuracy for identification of samples from HIT-positive patients than the SRA (AUC 0.93 vs. 0.82, respectively; p=0.01). When both sensitivity and specificity of an assay are important, one way to define a threshold for positivity is to determine the point on the ROC curve where the sum of sensitivity and specificity is maximized. This point in the PEA ROC curve corresponded to >24% p-selectin expression. For this analysis, a sample was considered PEA-positive if p-selectin expression was >24%, and inhibited >50% with HDH. Results are shown in Fig 1. The apparent superiority of the PEA over the SRA for identifying HIT-positive patients suggested that the PEA might be intrinsically more sensitive than the SRA for detecting platelet activating abs. This was confirmed by testing serial dilutions of 3 arbitrarily selected High-4T HIT abs against identical target platelets. A representative result is shown in Fig 2.The many attractive features of the PEA, especially its technical simplicity and its ability to identify patients judged to have a high likelihood of HIT suggest that its use can facilitate early diagnosis and improve management of this diagnostically challenging disorder.In Figures 1 and 2, the solid and dotted lines represent positive cut-offs of the SRA and PEA, respectively. [Display omitted] [Display omitted] [Display omitted] DisclosuresPadmanabhan:BloodCenter of Wisconsin: Patents & Royalties: A patent application has been filed based partly on these findings (Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591). Jones:BLOODCENTER OF WISCONSIN: Patents & Royalties: A patent application has been filed based partly on these findings (Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591). Bougie:BLOODCENTER OF WISCONSIN: Patents & Royalties: A patent application has been filed based partly on these findings (Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591). Aster:BLOODCENTER OF WISCONSIN: Patents & Royalties: A patent application has been filed based partly on these findings (Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591).

Development of a stringent ELISA protocol to evaluate anti-viral hemorrhagic septicemia virus-specific antibodies in olive flounder Paralichthys olivaceus with improved specificity.

Olive flounder were vaccinated with polyinosinic:polycytidylic acid [Poly (I:C)] to prevent viral hemorrhagic septicemia (VHS). Vaccine efficacy was verified by detection of anti- VHS virus (VHSV) antibodies using enzyme-linked immunosorbent assay (ELISA). In the study, ELISA absorbance values of the negative control group [Poly (I:C)-MEM10] were saturated when an ELISA protocol, that includes pretreatment of the fish sera with 5% skim milk, was used. However, the saturated OD values in the negative control did not correlate with a specific immune response against VHSV, because the group showed low survival rate (only 10%) following the VHSV challenge. Also, OD values of Poly (I:C)- VHSV group were high, and the group showed high survival rate (97.5%) against VHSV challenge test. It was suggested that the high OD values were possibly due to the presence of anti-fetal bovine serum (FBS) cross-reactivity. To compensate this, we subtracted the absorbance of infectious hematopoietic necrosis (IHNV)-Ag plates from those of the VHSV-Ag plates. However, the average value for the Poly (I:C)-VHSV group (0.167) was lower than expected even though high survival rate. We used an advanced ELISA system to pre-treat fish sera with 5% skim milk and two novirhabdoviruses as capture antigens as well as 50% FBS. The corrected absorbance values for pre-treated fish sera from the negative control Poly (I:C)-MEM10 and experimental Poly (I:C)-VHSV groups averaged 0.033 and 0.579, respectively. The specific VHSV antibody response of the vaccinated group was assessed using fish sera pretreated with skim milk and FBS and by calculating the corrected absorbance values from ELISA with two novirhabdovirus capture antigens.

P121 : HIGH NONSPECIFIC AT1R REACTION IN A NON-SENSITIZED KIDNEY RECIPIENT

Aim To investigate the origin of an extremely high AT1r result in a 0% PRA pediatric kidney recipient and history of nonspecific positive flow crossmatch. Methods Pt was HLA typed by rSSOP and 0% PRA was documented by LABScreen Single Antigen Assay (OL). Patient’s serum (P’s.S) was tested with AT1r ELISA (OL). The identical solid phase plate Maxisorp (Nunc) without the AT1r antigen was incubated with P’s.S and the kit Negative Control (NC). The plate was also treated with casein (Sigma) and BSA (Thermo Fisher), two reagents commonly used to block nonspecific reactions. A human serum (HS) sample with a negative AT1r reaction was used as a different source of protein. The secondary anti-IgG antibody conjugated with HRP and TMB from the AT1r kit was used to complete the reaction. Results Serum from a pediatric Pt with history of nonspecific flow crossmatch was used as part of the AT1r assay validation in our lab. Results with the undiluted serum were outside of the range as defined by the calibrator curve. Serial dilutions obtained an OD = 2034 and a concentration of 58.72 U/ml at titer 1:2048. The Pt was transplanted October 2013 with Thymo induction without any complications and maintains an excellent kidney graft function at the time of this abstract. With a history of no sensitization events and the graft outcome we suspect a nonspecific AT1r reaction. The P’s.S was incubated in an untreated plate to evaluate a positive reactivity against the polystyrene surface. We obtained an OD = 56 below the OD of the NC. When the P’s.S was added to the plate blocked with 0.5% casein in PBS the result was: P’s.S-OD = 3617/NC-OD = 43. The reading in the well blocked with 0.5% BSA in PBS was: P’s.S OD = 3071/NC-OD = 39. The reaction with the plate treated with HS (Neg: AT1r) was: P’s.S OD = 811/NC-OD = 777, both values are elevated but significantly lower than the well treated with the casein or BSA. Conclusions The lack of sensitization events, a favorable graft outcome, and these results lead us to believe that nonspecific reactions are to blame with regards to P’s.S reacting with the AT1r kit. Negative OD values with the untreated surface or treated with a HS versus high OD values with the surface treated with casein and BSA suggest a reaction against a protein of bovine origin present in the kit components. Further work will be needed to demonstrate this observation.

Transglutaminase 2: A Novel Autoantigen in Canine Idiopathic Central Nervous System Inflammatory Diseases

Necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE) and granulomatous meningoencephalomyelitis (GME) are common idiopathic inflammatory central nervous system (CNS) diseases with unknown etiology in dogs. We previously showed that IgG autoantibodies in the cerebrospinal fluid (CSF) of NME cases reacted to unknown brain proteins as well as to glial fibrillary acidic protein (GFAP). In the present report, we evaluated the autoantibodies against transglutaminase2 (TG2) in the canine CNS diseases. CSF samples obtained from dogs with NME (n=19), NLE (n=7), GME (n=11) and miscellaneous CNS diseases (n=12) were subjected. CSFs from 20 healthy dogs were used as controls. Indirect fluorescent antibody test on the canine cerebrum revealed astrocyte-binding IgG in the CSF of NME. After absorption of the CSF with bovine GFAP, the CSF still possessed the reactivity to astrocytes. Double-color staining showed clear colocalization of the autoantibodies and anti-human TG2 rabbit polyclonal IgG. An immunoblot assay against human recombinant TG2 revealed anti-TG2 IgG in the CSF from dogs with NME, NLE and GME. The CSF of canine idiopathic encephalitis cases, notably of NME, tended to show high ELISA OD values against human recombinant TG2 compared to healthy controls. The presence of anti-TG2 autoantibodies in the CSF may contribute to the elucidation of the etiology of canine NME, NLE and GME.

Testes de ELISA e vírus-neutralização na detecção de anticorpos contra o vírus da diarréia viral bovina no leite

O sucesso na estratégia de controle e erradicação do vírus da diarréia viral bovina (BVDV), passa necessariamente pela identificação e eliminação dos animais persistentemente infectados (PI). Como esses animais excretam continuamente o vírus em suas secreções e excreções, a prevalência de anticorpos no rebanho, frequentemente é alta e com altos títulos. Devido a essas características, amostras de tanques coletivos de leite, foram submetidas a duas técnicas sorológicas, a fim de estabelecer a mais adequada na realização de triagem de rebanhos. Para isso, 767 amostras coletivas de leite foram submetidas à análise por um kit ELISA indireto (teste referência) e pela técnica de vírus-neutralização (VNT) adaptada (teste proposto). Devido aos efeitos tóxicos do leite sobre o cultivo celular, a adaptação consistiu no aumento do volume final na etapa de incubação celular. Foram positivas, 177 e 139 amostras no ELISA e na VNT, respectivamente. Com isso, a VNT adaptada apresentou uma sensibilidade de 76,8% e uma especificidade de 99,5%. O índice Kappa (k) foi de 0,82, demonstrando uma ótima concordância entre as duas técnicas. A análise do coeficiente de correlação entre os valores de absorbância no ELISA (OD) e os títulos de anticorpos na VNT nas amostras positivas, demonstrou uma positividade moderada (r = 0,57) com p &lt; 0,05. No entanto, várias amostras com títulos altos na VNT apresentaram ODs moderadas ou baixas. Por outro lado, algumas amostras com títulos neutralizantes baixos apresentaram ODs altas. Como a presença de animais PI é sugerida por títulos neutralizantes ≥ 80, conclui-se que a técnica de VNT adaptada é mais adequada para a realização de triagem em amostras coletivas de leite quando objetiva-se detectar rebanhos com altos títulos de anticorpos.

Anti-Taenia solium larval stage Ig G antibodies in patients with epileptic seizures

Background:Cysticercosis is the most common differential diagnosis for epilepsy. The present study was carried out to assess the serological response among patients with epileptic seizures visiting JIPMER Hospital Puducherry.Materials and Methods:A total of 934 serum samples were collected from patients with epileptic seizures. A standardized questionnaire was designed to obtain information on the demographic, socioeconomic, environmental, and behavioral characteristics related to the transmission of infection. An enzyme-linked immunosorbent assay (ELISA) was used to detect the anti-Taenia solium larval stage IgG antibodies. Samples found reactive and inconclusive by ELISA were further tested by the enzyme immunotransfer blot (EITB).Results:The frequency of antibodies in the serum samples of the above-mentioned population was 16.2% by EITB. Anti-Taenia solium larval stage antibodies were detected in serum samples of 163 patients, out of which 27 (16.56%) patients belonged to the 0 – 15-year age group, 82 (50.30%) patients were in the 16 – 40-year age group, and 52 (31.90%) patients were above 41 years, respectively. Although the sera from males had higher OD values than those from females, the difference was not statistically significant. Out of 163 seropositive by ELISA, 152 (93.25%) were found to be positive by EITB. Out of the 152, 61 (40.13%) were farmers and 79 (51.97%) were office or factory workers.Conclusions:In conclusion, the results indicate a probable endemic situation and a high prevalence of cysticercosis in patients with epileptic seizures. Living in poor sanitary conditions seems to be an important factor related to human cysticercosis in Puducherry and the neighboring districts of Tamil Nadu.

Serological Survey of Encephalitozoon cuniculi Infection in Japanese Dogs

Antibodies to Encephalitozoon cuniculi were examined by enzyme-linked immunosorbent assay using E. cuniculi PTP2 recombinant protein and by Western blot analysis on a total of 472 dog serum samples that had been collected in Japan. Of these samples, 21.8% (103/472) had antibodies against E. cuniculi. Each of 5 serum samples that showed high (>1.0) or low (<0.1) OD value was selected randomly and further examined by Western blot using E. cuniculi-native antigens. All samples with high OD values reacted with specific E. cuniculi proteins, including an antigen of approximately 35 kDa corresponding with PTP2; sera with low OD values did not recognize this E. cuniculi band. This study is the first to demonstrate the prevalence of E. cuniculi infection in dogs in Japan.

B-CELL EPITOPE SPECIFICITY OF CARBOXY TERMINUS OF MYCOBACTERIUM PARATUBERCULOSIS ModD

Epitope mapping of ModD of Mycobacterium paratuberculosis was performed using overlapping peptides. In total, 80 overlapping peptides, covering the entire mature ModD, were commercially synthesized. Each peptide spanned 14 amino acids with an offset of 4 amino acids, i.e., with an overlap of 10 amino acids. Synthetic peptide antigenicity was evaluated by enzyme-linked immunosorbent assay (ELISA) using rabbit antisera to culture filtrate (CF) of M. avium or M. paratuberculosis or recombinant ModD (rModD). The peptides o f ModD reacting most strongly (ELISA OD > 1.0) were clustered near the N- and C-terminal ends. The peptides around the C-terminal end only showed the greatest specificity for M. paratuberculosis, yielding high ELISA OD values with rabbit anti-M. paratuberculosis CF serum and low ELISA OD values with rabbit anti-M. avium CF serum. Sera from naturally M. paratuberculosis-infected cattle, however, bound poorly to the short, 14-amino-acid peptides. Thus, two longer peptides covering amino acids 100 to 125 and 328 to 353 were synthesized based on their broad reactivity to rabbit serum against M. paratuberculosis CF. The peptide covering amino acids 328 to 353 showed the highest level of specific bovine antibody binding.

Indirect ELISA and Indirect Immunofluorescent Antibody Assay for Detecting the Antibody against Murine Norovirus S7 in Mice

To evaluate murine norovirus (MNV) infection in laboratory mice, we attempted to develop an enzyme-linked immunosorbent assay (ELISA) system and an indirect immunofluorescent antibody (IFA) assay for detecting the anti-MNV-S7 antibody in mice. MNV-S7, which was isolated in Japan, was used in both assays. The antigen for ELISA was prepared by ultracentrifugation of culture supernatants of RAW 264 cells infected with MNV-S7. Positive sera were obtained from 6-week-old, female C57BL/6JJcl mice inoculated orally with MNV-S7. IFA against infected RAW 264 cells was able to discriminate positive sera from negative sera. Indirect ELISA was performed using 96-well ELISA plates coated with formalin-treated MNV-S7 antigen. In this ELISA system, mouse sera obtained 2 weeks after infection or later showed significantly high OD values and were judged positive. An equal level of anti-MNV-S7 antibody response was observed in BALB/cAJcl, C57BL/6JJcl, DBA/2JJcl, and Jcl:ICR mice; whereas, C3H/HeJJcl mice demonstrated slightly lower antibody production 4 weeks after infection. We also used this ELISA system to evaluate 77 murine serum samples obtained from 15 conventional mouse rooms in research facilities in Japan and found that approximately half of the serum samples contained antibody to MNV-S7. We found that some serum samples were negative for antibodies to mouse hepatitis virus and Mycoplasma pulmonis but positive for antibody to MNV-S7. The results suggest that the MNV infection is more prevalent than other infections such as mouse hepatitis virus and Mycoplasma pulmonis in conventional mouse colonies in Japan, as is the case in other areas of the world.

Detection of dermcidin for sweat identification by real-time RT-PCR and ELISA

We evaluated the performance of real-time RT-PCR and ELISA assays for detection of dermcidin (DCD) in sweat and body-fluid stains. DCD, a small antibiotic peptide secreted into human sweat, was detected by real-time RT-PCR in 7-day-old stains containing as small as 10 μL of sweat, and the assay showed high specificity when testing 7-day-old stains containing 30 μL of other body-fluid. ELISA using anti-human dermcidin mouse monoclonal antibody detected DCD sweat diluted up to approximately 10,000-fold and could specifically detect DCD in 10 μL of body-fluid stains. The performance of the two assays was tested during winter on samples that simulated forensic case samples: an undershirt and a sock worn for 20 h, a handkerchief used to wipe the brow several times within 12 h, a cap and a cotton glove worn for 4 h, and a white robe worn at intervals for 2 years. The result showed that the former assay detected DCD in all sites of the undershirt examined (armpit, back, and breast), and the latter gave a relatively high OD value in the armpit among the three sites. For the socks, although the latter assay gave very high OD values in both the center and toe of the foot sole, the former could not detect DCD in both of them. These results indicate that highly damp conditions, such as inside a shoe, might promote the degradation of mRNA in samples such as socks. In the other case samples, sweat was adequately detected by both assays. This study is the first demonstration of the use of real-time RT-PCR to sensitively identify sweat among body-fluid stains, and it confirmed that dermcidin was an excellent marker for sweat identification. In addition, the usefulness of ELISA was also verified. Positive sweat identification using these assays is expected to assist forensic practice.

High-level expression of SAG1 and GRA7 gene of Toxoplasma gondii (Izatnagar isolate) and their application in serodiagnosis of goat toxoplasmosis

Toxoplasmosis, caused by Toxoplasma gondii, is a disease of economic importance in livestock, especially in sheep and goats, where it causes abortion. Although several serological tests are in use for diagnosis of infection, production of reliable reagents is a constraint. An 814 bp sequence coding for a truncated surface antigen surface antigen 1 (SAG1), a tachyzoite stage-specific protein, as well as a 657 bp sequence coding for granule protein 7 (GRA7), a dense granule protein were PCR amplified from the genomic DNA of T. gondii. The amplified products were ligated in pET-32b(+) and pET-32c(+) expression vectors, respectively and subsequently transformed into BL21(DE3)pLysS cells. A high-level expression of the histidine-tagged SAG1 and GRA7 fusion proteins were obtained after 7 h of incubation. The recombinant proteins were purified using Ni-NTA column and were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis using reference positive sera from goat, rabbit and humans at 1:100 dilution. Subsequently, the diagnostic efficiency of the recombinant proteins, either individually or as a cocktail of the recombinant proteins, was assessed with 56 reference goat sera by enzyme-linked immunosorbent assay (ELISA). The immunoreactivity of the refolded SAG1 and GRA7 was evidenced by high OD values. The reactivity of the recombinant proteins as a cocktail preparation was more than that of individual proteins in ELISA and could detect accurately the infection in goats. This is the first report of serological detection of caprine toxoplasmosis by ELISA using a cocktail of recombinant Toxoplasma proteins.

Detection of Specific Antibodies against Infectious Hematopoietic Necrosis Virus from Rainbow Trout Sera by ELISA using Two Novirhabdoviruses

An enzyme-linked immunosorbent assay (ELISA) with viral culture fluids as capture antigens was performed to detect specific antibodies against infectious hematopoietic necrosis virus (IHNV) from rainbow trout Oncorhynchus mykiss sera. When using IHNV antigen (IHNV-Ag), ELISA OD values for IHN-survived (IHN-Surv) rainbow trout sera were relatively higher than those for specific pathogen free (SPF) fish sera. However, some of the SPF sera were diagnosed as positive due to high OD values (> 0.2). To clarify reasons for these high OD values, each of five IHN-Surv and SPF sera was subjected to ELISA with viral hemorrhagic septicemia virus (VHSV) and hirame rhabdovirus (HIRRV) antigens (VHSV-Ag and HIRRV-Ag). Some of the IHN-Surv and SPF sera showed high OD values in both VHSV-Ag and HIRRV-Ag plates even though there was no possibility that those sera contained antibodies against VHSV or HIRRV antigen. These results suggest that some of the sera contained antibodies against impurities in viral culture fluids such as FBS and cell debris, and caused the false-positive reactions. The corrected OD values, subtracted the OD values in VHSV-Ag plates from those in IHNV-Ag plates, were all > 0.2, in SPF sera (n = 148) while those in IHN-Surv sera (n = 238) were randomly distributed from 0 to 0.7. It was considered that the corrected OD values may represent true values recognized by IHNV-specific antibodies.

Effects of Paratuberculosis on Productivity in Danish Dairy Cows

Paratuberculosis is a chronic enteritis in dairy cattle and other ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Clinical outbreak causes economic losses due to reduced milk production and slaughter weight and susceptibility to other diseases, and at herd level involuntary culling and death rate are increased. During the last twenty years it has become more and more obvious, that also subclinical paratuberculosis may have an effect on milk production, fertility, milk quality, susceptibility to mastitis, slaughter weight and culling rate. However previous studies of these effects on productivity offer conflicting results, which may be due to herd specific circumstances. In this study antibodies to MAP in milk samples from 7994 cows in 108 Danish herds were tested with enzyme-linked immunosorbent assay (ELISA). Readings were recorded on a continuous scale as the optical density (OD). These OD values were standardised according to parity and stage of lactation as previous studies on the same material show significant increase in ELISA response with increasing parity and in the first and last weeks of the lactation of infected cows. In order to use the diagnostic information in recordings on a continuous scale, subdivision into a positive and negative group by choosing a cut-off value is avoided. Response variables expressing productivity are: milk yield, milk quality (fat, protein and somatic cell count), days open, slaughter quality and weight. At herd level effects on death and culling rate will be analysed. Besides the primary explanatory variable: adjusted ELISA response to MAP, covariables such as age at first calving, parity, calving season, housing system and herd size are included in the analysis. Effects on productivity will be analysed with multi-level regression analyses. A preliminary analysis comparing the lowest10% with the highest 10%of the corrected OD-values indicate that average daily milk yield during the first 24 weeks of lactation, protein content of the milk, slaughter weight and quality are reduced in cows with high OD-values (probably infected cows) compared to cows with low OD-values (probably non-infected cows). More detailed analyses at cow and herd level will be presented at the 11th ICPD in August 2001.

Detection of antibodies to Borna disease virus (BDV) in Turkish horse sera using recombinant p40. Brief report.

The nucleoprotein of Borna disease virus (BDV-p40) was produced in a Baculovirus expression system using sf9 cells. The purity and specificity of the recombinant p40 was confirmed by SDS-PAGE and immunoblotting. The recombinant p40 was used in an ELISA to screen horse sera in Turkey. For this, 323 horses from selected cities in the Marmara region of Turkey were examined clinically and serum was collected from each. All horses were clinically healthy except for a few with wounds on the skin. Antibodies to BDV were detected in the sera of 82 (25%) of 323 horse sera. Six sera were selected that had low, medium or high OD values by ELISA and were analysed by Western blotting. All reacted specifically with p40 at a dilution of 1 in 1000. This is the first report of the detection of Borna disease in Turkey and needs further molecular biological investigations to compare the Turkish strains with those strains detected in Europe.

Experimental leprosy in monkeys. II. Longitudinal serological observations in sooty mangabey monkeys.

In this study, 11 SMM were grouped and inoculated with differing doses of SMM-origin Mycobacterium leprae (ML) between 4.5 x 10(8) and 1 x 10(9) by either combined IV/IC routes or by IV or IC route alone. The combined route was the most effective in eliciting progressive, disseminated LL leprosy. In all, 6 of 7 SMM inoculated by the combined routes developed leprosy requiring treatment at some point. Only 1 of 4 inoculated by a single route developed persisting leprosy requiring chemotherapy. Either no disease or spontaneous regression of initial disease occurred in the other 3 animals inoculated by a single route. Doses in excess of 1 x 10(9) ML were more effective than lesser doses. An association was observed between the development of IgG anti-PGL-I ELISA OD values and resistance to leprosy and between IgM anti-PGL-I and leprosy progression or susceptibility. Serum PGL-I antigen levels, determined by dot ELISA, paralleled disease severity longitudinally. High positive OD values of anti-LAM IgG prior to ML inoculation were observed in the majority of leprosy-susceptible SMM in contrast to negative levels in more resistant animals. Anti-LAM IgG OD values exceeded the positive cut-off point after inoculation in 5 of 11 SMM; 3 of these 5 had concurrent detectable serum levels of PGL-I antigen.