Indirect Enzyme-linked Immunosorbent Assay Kit Research Articles

Caprine arthritis and encephalitis virus infection in goats of Bangladesh: Serological detection and its associated risk factors.

Caprine arthritis and encephalitis (CAE) is a multisystemic persistent viral disease of goat that causes significant economic losses to the farmers and livestock sector. However, no information in this country is available regarding CAE virus (CAEV) infection. Therefore, this study aimed to estimate the seroprevalence of CAEV infection among the goat population in the selected goat-prone districts in Bangladesh and to identify the associated risk factors of the disease. From July 2021 to June 2022, 446 goat serum samples were randomly collected from the study area. Goat owners were interviewed using a pretested questionnaire to determine the risk factors. A commercial indirect enzyme-linked immunosorbent assay kit was used to screen blood serum for CAEV antibodies. Logistic regression models were used to analyze risk factors and serological data to identify the potential risk factors. Out of 446 serum samples, 19 samples were seropositive against CAEV. The overall seroprevalence was 4.26% (95% confidence interval [CI]: 2.58-6.57). The multivariable logistic regression model identified sex (Female; odds ratio [OR]: 3.98; 95% CI: 1.13-13.95), animal age (12-48 months; OR: 4.93, 95% CI: 0.63-38.13), and biosecurity status (Poor biosecurity; OR: 1.66, 95% CI: 0.46-5.92) as potential risk factors for CAEV seropositivity. This study revealed the serological detection of CAEV in Bangladeshi goats where seroprevalence is found to be relatively low. To eradicate the disease, screening and culling of infected goats from the herd might be implemented.

Validation of a Commercial Indirect ELISA Kit for the Detection of Bovine alphaherpesvirus1 (BoHV-1)-Specific Glycoprotein E Antibodies in Bulk Milk Samples of Dairy Cows.

Simple SummaryRecent studies have assessed the feasibility of using commercial indirect enzyme-linked immunosorbent assays (ELISA) on bulk milk (BM) samples. Today is a need to validate a diagnostic method for granting and maintaining a correct surveillance strategy for infectious bovine rhinotracheitis (IBR). However, the poor availability of reference materials for diagnostic tests on milk samples and, the low sensitivity of blocking glycoprotein E (gE) ELISAs using milk as a matrix, represent limitations for developing new assays to control infectious diseases in livestock. This study aimed to validate a commercial indirect ELISA kit to detect antibodies to gE of Bovine alphaherpesvirus 1 (BoHV-1) from BM samples, according to the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The samples were collected from an IBR outbreak. The findings demonstrated high diagnostic performances. Furthermore, the validated kit is an easy-to-use and economical method to discriminate between BoHV-1 infected or immunised animals with gE-deleted markers vaccines using BM samples. Moreover, milk sampling represents a fast and non-invasive procedure for animals because it avoids negative effects, such as stress, and reduces the total cost of surveillance. In this study, we validated a commercial indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies to glycoprotein E (gE) of Bovine alphaherpesvirus 1 (BoHV-1) in bulk milk (BM) samples using the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The assay performance characteristics were evaluated using a panel of positive (n = 36) and negative (n = 80) samples with known infectious bovine rhinotracheitis (IBR) status. The assay showed adequate repeatability (within-run and between-run), with a coefficient of variability (CV%) of replicates below 30%; only two 1:40 diluted samples had a CV% above 20%. Additionally, an agreement analysis of the qualitative results of replicates led to a Gwet’s agreement coefficient of 0.99 (95% confidence interval (CI): 0.96–1.00, p < 0.001). The estimated diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 100% (95% CI: 90.3–100%) and 97.5% (95% CI: 91.3–99.7%), respectively. Overall, a good level of agreement was observed between the assay results and the true IBR status of samples (weighted Cohen’s κ: 0.96, 95% CI: 0.78–1.00). The findings demonstrate that the indirect ELISA kit validated here is an easy-to-use and economical method to differentiate infected and gE-deleted marker vaccine-immunised animals using BM samples.

Seropositivity and risk factors associated with the presentation of bovine leukosis virus in Sotaquirá, Colombia.

Background and Aim:Enzootic bovine leukosis is a disease economically important to the dairy farming industry worldwide. The virus is of the Deltaretrovirus genus and is primarily transmitted iatrogenically. Most bovines infected with the virus remain asymptomatic with only 5-10% of cattle having lymphomas. This study aimed to determine the seroprevalence of bovine leukosis virus (BLV) in Sotaquirá, Boyacá, Colombia.Materials and Methods:We conducted a descriptive, observational epidemiological cross-sectional study using the simple random sampling method with a sample size of 1000. Blood samples from random bovine were processed using the SERELISA® BLV Ab Mono Blocking indirect enzyme-linked immunosorbent assay kit (Zoetis, USA). The assay had a sensitivity of 97% and a specificity of 98%. The collected data were processed using Epi Info® (Centers for Disease Control and Prevention; Atlanta, Georgia). From the study, we could determine a high seroprevalence of BLV in Sotaquirá.Results:We established a high seroprevalence on BLV in the municipality, with 31.1% apparent seroprevalence and 30.6% real seroprevalence rate. We found that male cattle more than 4 years old (39.4%) and the Ayrshire breed (45.5%) had the highest prevalence rates of the virus. In this study, we could establish statistically significant associations according to breed, age, and gender of the cattle under study. Moreover, we identified the risk factors for BLV infection. We found that in cattle aged <1 year and those older than 4 years of age and those of the Holstein breed, the presentation of infectious bovine rhinotracheitis, mucosal secretions, mastitis, fetal death, the presence of a corral, and the implementation of artificial insemination practices were risk factors for BLV infection.Conclusion:Determining the prevalence of BLV within the herd and identifying the associated risk factors for the disease are fundamental in developing efficient programs for the control and eradication of BLV within herds.

Detection of anti-HEV antibodies and RNA of HEV in pigs from a hyperendemic Italian region with high human seroprevalence.

Pigs are considered the main reservoir of genotypes 3 and 4 of hepatitis E virus (HEV), which is the major cause of acute hepatitis of viral origin in humans worldwide. An increasing number of autochthonous HEV infections have been observed in recent years in industrialized countries, most likely as a result of zoonotic transmission through the consumption of raw or undercooked meat products. Two hundred and thirty-three blood and liver samples were collected at four different local slaughterhouses from domestic pigs bred in Abruzzo, a region of south-central Italy, where there is the highest human seroprevalence to HEV compared with the rest of Italy. An indirect enzyme-linked immunosorbent assay kit was used for detecting anti-HEV IgG in the sera, while the presence of HEV RNA was investigated by performing a real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Between 87.3% and 100% of swine serum samples collected in different slaughterhouses of Abruzzo were positive for anti-HEV antibodies. Conversely, none of the liver samples collected from the same animals were positive for HEV by real-time RT-PCR. The hypothesis of foodborne zoonotic transmission from local pigs as responsible for the hyperendemic status of Abruzzo cannot be corroborated. However, the high seroprevalence observed in pigs indicates that HEV is highly circulating in these territories. We propose to further investigate the role of wild fauna and trade in carrier pigs, and the maintenance of HEV virulence in the environment and meat supply chain to shed light on the possible sources of human infection and the degree of occupational risk.

Seroprevalence and risk factors of bovine respiratory syncytial virus in cattle in the Nineveh Governorate, Iraq.

Background and Aim:Bovine respiratory syncytial virus (BRSV) is one of the main causes of severe pneumonia, interstitial edema, and emphysema in cattle. The current study investigated the prevalence and risk factors of BRSV in cattle in the Nineveh Province, Iraq.Materials and Methods:Between September 2017 and September 2018, 450 serum samples were collected from non-vaccinated cattle of different ages and breeds for BRSV testing. The epidemiological information of the animals was recorded. The prevalence of the disease was determined using an indirect enzyme-linked immunosorbent assay kit.Results:The prevalence of BRSV was 83.11%, and it was significantly (p<0.05) higher in cattle aged greater than 7 months-1.5 years than in older animals; in imported cattle than in Native animals; and in animals originating from large herds (100 animals) than in those from smaller herds (40 animals). There was no significant difference between BRSV prevalence in male and female animals. When samples from different regions of the Nineveh Governorate were compared, the northern region was associated with the highest prevalence of the disease. Samples harvested in the winter displayed the highest BRSV titer, compared to those collected during the other seasons.Conclusion:BRSV is prevalent in cattle from the Nineveh Governorate. Risk factors such as animal age, origin, herd size, and the herd’s geographical location are associated with an increased prevalence of the disease in this region. Routine vaccination programs should be adopted to reduce the prevalence of BRSV.

Polyamic acid (PAA) immobilized on glassy carbon electrode (GCE) as an electrochemical platform for the sensing of tuberculosis (TB) antibodies and hydrogen peroxide determination

Tuberculosis (TB) is a chronic infectious disease that usually affects the pulmonary region of human beings. It remains critically important to develop cost effective methods for TB detection, especially since impoverished communities are often the target population. Immunological assays are commonly used for the detection of TB and are typically based on selected antibody (Ab) antigen (Ag) interactions as measured by the enzyme-linked immunosorbent assay (ELISA). It is also common to have tagged antibody/antigen binding pairs where the signaling is produced by the tag as is the case with the indirect tuberculosis IgG ELISA Kit, where a horseradish peroxidase (HRP) tag on the antibody is responsible for the analytical signal. Mycobacterium tuberculosis (MTB), recombinant protein antigen 85B (Ag85B), is the most abundant protein exposed by Mycobacterium tuberculosis acting as a potent immuno-protective antigen. In this work, both Ag85B and antibody-HRP (Ab-HRP) were immobilized on a semi-conductive polyamic acid (PAA) electrochemical interface towards the design of new electrochemical sensors. In a first approach, the antigen, Ag85B, was employed as the bioreceptor to afford label-free TB signaling through antibody detection and the obtained limit of detection was 8 μg/mL of antibody. The response was evaluated by voltammetric methods and electron impedance spectroscopy (EIS). Cyclic voltammetry (CV) and EIS were used to characterize the charge transfer and capacitive behavior of the label-free sensor. In a second approach, the HRP tagged antibody was used for the design of a stable hydrogen peroxide (H2O2) sensor with very good analytical performance with a limit of detection equal to 1.3 μM. Even though we developed an ultrasensitive H2O2 sensor, no further development of TB antigen detection was possible in this work.

Recombinant DENV 2 NS5: An effective antigen for diagnosis of DENV infection

Dengue fever is a mosquito-borne viral disease with dramatically increasing morbidity rate worldwide in decades. Since there is no specific treatment to date, early diagnosis is important for providing proper timely medical care to minimize mortality, and for the prompt initiation of public health control measures. NS5 is a potential biomarker for dengue virus infection due to its highly conserved and immunogenic properties. In this study, the DENV 2 NS5 full-length and the DENV 2 NS5 C-terminus RNA-dependent RNA polymerase domain fragment (NS5-C70) expression plasmids were constructed, and the 104 kDa full-length NS5 and the 70 kDa NS5-C70 were respectively expressed in Escherichia coli. These two purified recombinant products were found to react with the sera of patients infected with dengue virus when analyzed by an enzyme-linked immunosorbent assay (ELISA), which resulted in significantly higher absorption values than those of control sera. The recombinant DENV 2 NS5 exhibited strong reactivity to each of the four types of sera, whereas the NS5-C70 showed strong reactivity only to DENV 2 and 4. In comparison, the positive agreement value of recombinant NS5-based assay with either MyBioSource or Panbio assay was higher than that of the two commercially available IgG indirect ELISA kits. These results suggest that the recombinant DENV 2 NS5 be an effective antigen for detection of dengue virus infection. The recombinant NS5-C70 may also be used as an auxiliary antigen for diagnostic purposes.

Biotechnology for obtaining hybrid positive control samples for immunoassay for detecting antibodies against Chlamydia trachomatis

The enzyme-linked immunosorbent assay (ELISA) is the most informative and versatile method of serological diagnostics. The possibility of detection by ELISA of specific antibodies of different classes allow one to differentiate primary infectious processes and their remission, exacerbation and chronic disease (holding of differential diagnosis). This approach is implemented in the methodology for evaluation of patients for the presence of humoral immune response against TORCH-infections pathogens (toxoplasmosis, rubella, cytomegalovirus, herpes simplex viruses infections, and some others). Therefore, testing for the presence of specific IgG and IgM antibodies against TORCH-infection pathogens in blood serum is an important element of mother and child protection. The essential problem in the production of IgM-capture ELISA diagnostic kits is obtaining positive control. The classic version of positive control is human blood serum (plasma) containing specific antibodies. But specific IgM-positive sera are insignificant raw material. This fact can significantly limit the production of diagnostic kits, especially in case of large-scale manufacture. We have suggested a methodological approach to the use of synthetic positive controls in IgM-capture ELISA kits based on conjugate of normal human IgM and monoclonal antibodies against horseradish peroxidase. It was found that it is possible to realize such a task by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), reductive amination-mediated conjugation (by sodium periodate) and glutaraldehyde-mediated conjugation. It was found that conjugates of normal human IgM and monoclonal antibodies against horseradish peroxidase obtained using NHS ester-mediated maleimide conjugation and periodate method are homogeneous in molecular weight, whereas conjugate synthesized by glutaraldehyde method comprises at least three types of biopolymers with close molecular weight. It was found that synthetic positive control obtained by different methods are characterized by higher titre compared to IgM-positive high-titre serum. However, positive control obtained by NHS ester-mediated maleimide conjugation has the best titration profile characteristics. We have suggested a methodological approach to the use of synthetic positive controls in indirect ELISA kits based on conjugate of normal human IgM (IgA) and monoclonal antibodies against major outer membrane protein of Ch. trachomatis. It was found that it is possible to realize such task by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and reductive amination-mediated conjugation (by sodium periodate). It was found that synthetic positive control obtained by different methods are characterized by higher titre compared to IgM- and IgA-positive high-titre serum. However, positive control obtained by NHS ester-mediated maleimide conjugation has the best titration profile characteristics, both at the release time and after a week’s storage at 37 °C.

Decline of antibody response in indirect ELISA tests during the periparturient period caused diagnostic gaps in Coxiella burnetii and BVDV serology in pluriparous cows within a Holstein dairy herd

In cattle, indirect enzyme-linked immunosorbent assay (ELISA) tests are commonly used in serological routine diagnostics. In a longitudinal study design, changes in relative optical density (OD) from drying-off until week 11 after calving were analyzed in blood and milk samples from pluriparous dairy cows (n=21) using a commercial indirect anti-C. burnetii ELISA test. In a second part of this study, changes over time were evaluated in blood and milk samples of 11 of these cows in an indirect ELISA detecting anti-Bovine viral diarrhea virus (BVDV) antibodies. Regarding the cows which were still in the herd at the time of calving, blood serological qualitative changes from positive to negative or indeterminate were demonstrated in 7/20 cows (35%) for the anti-C. burnetii indirect ELISA and in 2/10 cows (20%) for the anti-BVDV indirect ELISA, respectively, during the period from 14days ante partum to calving. Relative OD in the anti-C. burnetii and the anti-BVDV indirect ELISA tests followed basically similar courses over time. In blood serum, relative OD initially increased after drying-off, before a drop around calving was observed. After the colostrum period, relative OD in blood serum showed an increase until week 11 of lactation. In colostrum samples, relative OD levels were higher than in milk samples obtained one day before drying-off. After parturition, relative OD in milk decreased until week 6 of lactation in the anti-C. burnetii indirect ELISA and until week 11 in the anti-BVDV indirect ELISA, respectively. In conclusion, blood serological investigations in periparturient dairy cows using indirect ELISA kits should be avoided.

Sensitivity and specificity of indirect ELISA for the detection of antibody titers against BVDV from beef cattle raised in Pará State

The aims of this study were to establish the prevalence of anti-bovine viral diarrhea virus (BVDV) antibodies (Ab) in beef cattle raised in Pará state, to compare the prevalence of seropositive animals to BVDV using a commercial indirect enzyme-linked immunosorbent assay kit (iELISA) and the virus neutralization (VN) test, and finally, to determine the sensitivity (Se) and specificity (Sp) of the iELISA for the detection of anti-BVDV Ab using VN as a gold standard. A total of 400 serum blood samples from Nelore cows aged at least 24 months from five farms in the Pará state from two mesoregions (Metropolitan Region of Belem and Northeast of Pará) were analyzed. All animals were vaccinated against brucellosis and foot-and-mouth disease. The examination of anti-BVDV Ab with VN was performed in the Laboratory of Bovine Viruses of the Biological Institute of Sao Paulo as described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. For VN, bovine kidney epithelial cells from the Madin Darby Bovine Kidney (MDBK) strain were used. The determinations of anti-BVDV Ab were performed with the iELISA test at the Laboratory of Immunology and Microbiology of the Federal Rural University of Amazonia according to the manufacturer's recommendations. The results were classified as follows: (a) correct positive diagnosis, (b) incorrect positive diagnosis, (c) correct negative diagnosis, and (d) incorrect negative diagnosis, according to the results obtained from VN. From the values obtained from VN and iELISA, Se [(a ÷ a + d) × 100], Sp [(c ÷ c + b) × 100], positive predictive value [(a ÷ a + B) × 100], and negative predictive value [(c ÷ c + d) × 100] were calculated for iELISA. The frequencies (%) of seropositive animals were determined and compared both between the different tests (iELISA and VN) and between the different farms (1, 2, 3, 4, and 5). The statistical analysis was performed with a significance level of 5%. The prevalence of seropositive animals was found to be different (P &lt; 0.0001) using VN (39.25% [157/400]) and iELISA (54.50% [218/400]). It was observed that the Se and Sp of the iELISA assay were 98.72% and 74.07%, respectively. Of the total, 25.93% (63/243) of the samples were considered false-positive and 1.27% false-negative (2/157). It was concluded that the BVDV infection is present in beef cattle herds of the state of Para. Based on the speed of execution, ease of handling, and high Se of the iELISA, it is suggested that this assay can be used as a screening test for the detection of anti-BVDV Ab with the aim of eliminating infected animals from large herds of beef cattle.

English

Very little information is available in Algeria on Q fever and chlamydial abortion sheep, two zoonosis caused by Coxiella burnetii and Chlamydophila abortus and their main reservoirs are domestic ruminants.&nbsp; This study aimed at investigating the seroprevalence of these two diseases in sheep flocks from six Da&iuml;ra (Telagh, Tanira, Moulay Slissen, Marhoum, Ras Elma and Merine). A serological survey was conducted in 39 flocks with a history of abortions, which were classified by size. A total 180 sera were collected from the aborted ewes. Q fever indirect ELISA kit and C. abortus indirect ELISA kit (ID Screen&reg;) kits were used to know the percent prevalence in sheep. The results showed that 28% (N = 50/180) of sheep were seropositive for Q fever and 31% (N = 55/180) of sheep were seropositive for chlamydial abortion. Twenty eight herds (72%) showed at least one seropositive animal for Q fever and 29 herds (74%) showed at least one seropositive animal for chlamydial abortion. Larger herds led to more infected herds of small and medium for Q fever. These results showed that infection with Q fever and chlamydial abortion were common in the study area, therefore encouraging efforts are needed to propose measures to reduce the spread and zoonotic risk. Key words: Q fever, chlamydial abortion, seroprevalence, enzyme-linked immunosorbent assay (ELISA), zoonosis.

Molecular and serological detection of Neospora caninum in multiple tissues and CSF in asymptomatic infected stray dogs.

Backgrounds: Neospora caninum is an Apicomplexan parasite. It causes paralysis and neuromuscular disorders in dogs and abortion in cattle. Although contamination with N. caninum is common in stray dogs, most of the dogs are infected with subclinical neosporosis. Objectives: The objective of this study was to evaluate the presence of N. caninum in multiple tissues and cerebrospinal fluid (CSF) using Nested-PCR technique. Furthermore, the N. caninum specific antibody was detected in serum of examined dogs by indirect enzyme-linked immunosorbent assay (ELISA). Methods: Forty two stray dogs of mixed breeds captured from districts of Tehran, Iran, were examined physically and euthanized. A commercially indirect ELISA kit was used to detect the anti-N. caninum antibodies in serum. Nested PCR was applied to analyze the extracted DNA from brain, skeletal muscle, CSF, liver, spleen and mandibular lymph nodes for Nc5 gene. Results: Indirect ELISA assay for N. caninum antibody was positive in a seven years old male dog (2.22%). Out of 42 stray dogs whose multiple organs were examined using Nested PCR, 15 samples (35%) were positive. The highest presence of N. caninum was found in skeletal muscle with 30% (13/42) frequency, followed by CSF (26.2%) (11/42), brain (19%) (8/42), liver (7.14%) (3/42), lymph node (4.62%) (2/42) and spleen samples (0/42). Conclusions: These results suggest that serology tests can underestimate the carrier of N. caninum in dogs compared to PCR test which directly detect the presence of the parasite. Nested-PCR might be a potential alternative for the histological methods to detect N. caninum in dog’s tissues.

Selected metabolic biochemical and enzyme activities associated with Besnoitia besnoiti infection in dairy cattle.

The main aim of the study was to explore, compare, and identify whether there is an association between Besnoitia besnoiti seropositivity in apparently healthy dairy cows with some biochemical parameters, enzyme activities, and beta-hydroxybutyrate (BHBA). A total of 98 dairy cows were included in the study, of which there was 50 seropositive and 48 seronegative cows. Analysis of serum antibodies against B. besnoiti antibodies was performed using an indirect enzyme-linked immunosorbent assay kit. Student's independent t test showed that there was a significant difference in BHBA, albumin, and lactate dehydrogenase (LDH) between the seropositive and seronegative groups. Univariable regression analysis showed no significant association between seropositivity status with any of the evaluated parameters except BHBA level, mastitis, and abomasum displacement. Multivariable logistic regression analysis showed that there was a strong association between seropositivity with BHBA level. The significant association between BHBA and B. besnoiti seropositivity represents preliminary finding that needs further exploration.

Coxiella burnetii(Q Fever) Infection in Farmed Ruminants in Three Northeastern Provinces and Inner Mongolia Autonomous Region, China

Coxiella burnetii is the pathogenic agent of Q fever, a zoonosis that occurs worldwide. In China, the organism infects people and a wide range of wild and farmed animals. However, little is known about C. burnetii infection in farmed ruminants in northeastern China, and there appears to have been no previous study on sika deer. The present study was conducted to determine the seroprevalence of C. burnetii in farmed ruminants in this region of China. A total of 3147 domestic ruminants (784 sheep, 1016 cattle, and 1347 sika deer) were randomly collected for this study. An indirect enzyme-linked immunosorbent assay kit was used to detect the antibodies to C. burnetii. The results showed an overall seroprevalence of 16.7% in domestic ruminants (24.9%, 13.5%, and 12.3% in cattle, sheep, and sika deer, respectively). This is the first report of C. burnetii seroprevalence in sika deer in China. Protective and hygiene measures should be applied in farm management to reduce the risk of exposure of C. burnetii to humans.

Synthetic positive controls for ELISA test kits for detection of IgA and IgM antibodies to Chlamydia trachomatis

The enzyme-linked immunosorbent assay (ELISA) is the most informative and versatile method of serological diagnostics. The possibility of detecting by ELISA specific antibodies of different classes allow to differentiate primary infectious process and its remission, exacerbation and chronic disease (holding of differential diagnosis). This approach is implemented in the methodology for evaluation of patients for presence of humoral immune response against the causative agent of urogenital chlamydiosis. As with other infections immediately after Chlamydia trachomatis infection the specific IgM antibodies are formed, and subsequently basic projective antibodies of IgG class are synthesized. However, at exacerbation of chronic urogenital chlamydiosis specific IgA antibodies can be synthesized. That is why comprehensive evaluation of patients for presence of humoral immune response to Ch. trachomatis involves plasma testing of specific antibodies of all three classes. The essential problem in the production of ELISA diagnostic kits is obtaining of positive control. The classic version of positive control is human blood plasma containing specific antibodies. But specific IgM- and IgA-positive sera are deficit raw materials. This fact can significantly limit the production of diagnostic kits, especially in case of large-scale manufacture. We have suggested methodological approach to use of synthetic positive controls in indirect ELISA kits based on conjugate of normal human IgM (IgA) and monoclonal antibodies against major outer membrane protein of Ch. trachomatis. It was found that it’s possible to realize such task by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and reductive amination-mediated conjugation (by sodium periodate). It was found that synthetic positive controls obtained by different methods are characterized by higher titer compared to IgM- and IgA-positive high-titer serum. However, positive control obtained by NHS ester-mediated maleimide conjugation has the best titration profile characteristics, at the release time and after one-week storage at37 °C.

Some newer marker phytoconstituents in methanolic extract of Moringa oleifera leaves and evaluation of its immunomodulatory and splenocytes proliferation potential in rats.

Objectives:The present study was undertaken to unravel the newer marker phytoconstituents in methanolic extract of Moringa oleifera leaves (MOLE) and evaluation of its immunomodulatory and splenocytes proliferation potential in rats.Materials and Methods:Hot methanolic extract of MOLE was subjected to gas chromatography-mass spectrometry (GC-MS) analysis. Immunomodulatory potential was studied in four groups of rats following administration of MOLE at 62.5 and 125 mg/kg for 21 days, followed by immunization with Salmonella typhimurium “O” antigen and antibody titer determined using indirect enzyme-linked immunosorbent assay kit. Total lymphocytes and T- and B-lymphocytes count were determined in control and after MOLE administration (62.5 and 125 mg/kg) to rats for 42 days. Splenocytes (2 × 106 spleen cells/ml) from MOLE treated rats were harvested and stimulated using concanavalin A and optical density (OD) and stimulation index were determined. Splenocytes from healthy control rats were also collected and treated in vitro with different concentrations of MOLE (5, 10, 25, 50, and 100 µg/ml) and concanavalin A to determine effect of MOLE on OD and stimulation index.Results:GC-MS analysis revealed presence of 9,12,15-octadecatrienoic acid ethyl ester, 6-octadecenoic acid, cis-vaccenic acid and 2-octyl-cyclopropaneoctanal in MOLE. MOLE at 125 mg/kg increased the antibody titer by 50%. Although there was slight decline in lymphocytes count (total, B- and T-lymphocytes) in MOLE treated rats, percentage of T-lymphocytes was increased nonsignificantly. Ex vivo and in vitro studies revealed marked increase in OD and stimulation index indicating MOLE-induced splenocytes proliferation.Conclusion:GC-MS study revealed four new compounds in MOLE apart from promising its immunomodulatory potential based on humoral immune response, percentage increase in T-lymphocytes count, and induction of splenocytes proliferation.

Comparitive study on the efficacy of ELISA and IS900 PCR for the diagnosis of Paratuberculosis in goats

Paratuberculosis, one of the chronic granulomatous enteritis that predominantly affects ruminants worldwide, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is most efficiently diagnosed by MAP from faeces by Polymerase Chain Reaction (PCR). Serological tests like Enzyme Linked Immuno Sorbent Assay (ELISA) also provides a rapid and cost-effective alternative diagnostic tool. Present study was carried out to directly evaluate the sensitivity and specificity of ELISA (ID vet innovative diagnostics; France) using IS900 PCR as a gold standard. Serum and faecal samples were collected from 180 adult goats of either sex, from Malappuram and Thrissur districts of Kerala with unknown paratuberculosis status. Faecal samples were processed for direct IS900 PCR and serum samples were tested for MAP antibodies using Indirect ELISA kit. IS900 PCR detected 38 out of 180 confirmed to be shedding MAP. ELISA detected 22 out of 180 animals as positive. Overall, ELISA was 50 % sensitive and 97.9 % specific in comparison to IS900 PCR. The IS900 PCR outperformed ELISA in detecting animals potentially infected with MAP and is more sensitive than ELISA at detecting animals suspected of paratuberculosis. But, for early diagnosis of paratuberculosis in goats, ELISA can be done as easy and rapid farm level identification and IS900 PCR as individual confirmatory test.

Seroprevalence and risk factors associated with Chlamydophila spp. infection in ewes in the northeast of Algeria

A cross-sectional study was carried out to estimate prevalence of Chlamydophila spp. antibodies and to investigate risk factors associated with chlamydial infection in 552 ewes between March 2011 and January 2012 in the province of Constantine. Anti-Chlamydophila antibodies were detected using an indirect enzyme-linked immunosorbent assay kit in 24.5% of examined sera. Of the herds, 70.4% had at least one seropositive animal. A pretested structured questionnaire was administered in order to collect information on individual animal health and herd management practices. Chi-square analysis and multivariable logistic regression model were used to identify risk factors related to Chlamydophila seropositivity. Univariable analysis revealed 17 variables with p < 0.25 that were offered to the multivariable logistic regression model which in turn identified 12-23 months age group (OR = 5.903, 95% CI (OR) = 1.690; 20.618) and not using disinfectants (OR = 2.099, 95% CI (OR) = 1.314; 8.065) as risk factors for Chlamydophila spp. seropositivity. Moreover, occurrence of stillbirth problem (OR = 3.682, 95% CI (OR) = 1.825; 7.430) and 5-10% mortality rate in young lambs (OR = 2.584, 95% CI (OR) = 1.058; 6.310) were significantly associated with seropositivity to Chlamydophila spp. On the other hand, availability of veterinary service was identified as a protective factor (OR = 0.161, 95% CI (OR) = 0.051; 0.511).

Comparative Performance of In-House and Commercially Available ELISA Kits for the Detection of Antibody Against Newcastle Disease Virus Vaccine

An In-house Indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibody titre against Newcastle disease virus (NDV) and was compared its sensitivity and specificity with the commercially available NDV antibody detection ELISA kit (Biocheck®, USA). The reference NDV was purified by centrifugation, ultracentrifugation and by sucrose density gradient ultracentrifugation. This purified NDV was used for coating of 96-well flat bottomed microtitre plate and to raise hyperimmune sera (known) in Fayoumi chickens. In the standardization test, the antigen dilution of 10-6 and the serum dilution of 10-3 were considered to be optimum for the present ELISA system. The correlation regression analysis was performed to construct a standard curve equation where a good positive correlation was observed (r = 0.912, n = 8, P&lt;0.01). The equation was used to convert corrected absorbance readings of the single working dilution (1 : 1000) directly into predicted ELISA antibody activity titres. In the sensitivity and specificity test, the serum dilution of 10-5 appeared to be the highest dilution which had the maximum lowest capacity to bind with the coated antigen of the present ELISA kit and only anti-NDV serum was found to bind with the coated antigen instead of serum of IBDV in the plate which revealed the high specificity of the developed In-house Indirect ELISA kit. In a comparative study with the 80 chicken sera samples, a significant positive correlation (r = 0.901, n = 80, P&lt;0.01) was found between In-house Indirect ELISA and commercial ELISA kit (Biocheck®, USA).DOI: http://dx.doi.org/10.3329/pa.v22i1-2.16467 Progress. Agric. 22(1 &amp; 2): 55 - 64, 2011

Expression of &lt;I&gt;OmpK&lt;/I&gt; gene of &lt;I&gt;Vibrio alginolyticus&lt;/I&gt; and development of a detection method of indirect ELISA

Vibrio alginolyticus is the main Vibrio pathogen in aquaculture in the south of China,the number of which is the largest in marine Vibrio class.In the present study,the outer membrane protein K gene(OmpK)of V.alginolyticus strain ATCC17749 was amplified by PCR and cloned into high efficient expression vector pET28a.The results of sequencing and restriction enzyme analysis combined with agarose gel electrophoresis showed that the open reading frame sequence was correct and fully consistent with what had reported.The recombinant plasmid of pET-28a-OmpK was successfully constructed and transformed into E.coli BL21 pLys E.The fusion protein was expressed under the IPTG inducing condition,and the expression product was purified by an affinity chromatographic method.Mouse anti-OmpK poly-clonal antibodies(PAbs)were obtained via applying protein OmpK as antigen to immune mice.Western-blotting analysis proved the recombinant protein has a good reactive ability against OmpK positive serum.Then,an indirect Enzyme-Linked Immunosorbent Assay(ELISA)for the rapid diagnosis of V.alginolyticus has been developed using the PAbs.The lowest V.alginolyticus suspension was 104 CFU/mL.Cross reactions of antisera with other bacteria were detected,and all results were negative.The results indicated the suitability and simplicity of the test as a rapid,field diagnostic tool for V.alginolyticus and it can be used for the rapid detection of V.alginolyticus.Further investigation will be focused on the development of an indirect ELISA Kit for the detection of V.alginolyticus.