Competitive Enzyme Linked Immuno Sorbent Research Articles

Seroprevalence and risk factors for brucellosis in cattle and rodents in Kilosa district, Morogoro, Tanzania

Background: Brucellosis is a global zoonosis caused by gram-negative bacteria that affects a diverse array of hosts including humans, domestic animals as well as wild animals such as cattle (B. abortus), goats, and sheep (B. melitensis), pigs (B. suis), and rodents (B. neotomae), and results in financial setbacks in the livestock industry. This study aimed to identify risk factors and estimate the seroprevalence of brucellosis in cattle and rodents in Kilosa district, Tanzania. Methods: A cross-sectional study was conducted from January 2023 to March 2023, cattle were randomly selected and rodents were trapped using Sherman, wire cages, and havahart traps. Blood samples were collected from the jugular vein and heart of the cattle and rodents, respectively. Sera were harvested from the collected blood and stored at – 20 ºC. All the sera were screened for brucella antibodies using the Rose Bengal Plate Test (RBPT) and confirmed by competitive Enzyme Linked Immuno Sorbent Assay (c-ELISA). The risk factors were captured using a structured questionnaire and analyzed by computing the Chi-square test and generalized linear model. Results: The seroprevalence of brucellosis was found to be 5.31% in cattle (95% CI: 0.0286-0.089) and 0.72% in rodents (95% CI: 0.0002-0.0397). A significant association was observed between grazing style and brucellosis seropositivity in cattle, with cattle that grazed together with sheep and goats having significantly higher odds of seropositivity (OR=6.5; 95% CI: 1.74-42.17, **p < 0.01). Conclusion: The detection of Brucella antibodies in both species indicates ongoing transmission and potential risk to public health. Our findings suggest that rodents may serve as reservoirs of brucellosis, contributing to its persistence and spread. Further research is essential to characterize the specific brucella species circulating among cattle and rodents and to understand the dynamics of interspecies transmission.

Sources and Prevalence of Aflatoxin B1 in Different Rice Paddies of Punjab and Sindh, Pakistan

Rice is a major cash crop used all over world. The hygroscopic nature of rice kernel with warm and fumic conditions is favorable to enhance the growth and development of toxigenic fungi which produce mycotoxins. This study estimates the prevalence of aflatoxin B1 in different rice paddies collected from twelve regions of the Punjab and Sindh provinces of Pakistan. All the samples were analyzed for their phenotypic characteristics (appearance, odor, and grain length). Competitive Enzyme Linked Immuno Sorbent Assay (ELISA) was used to evaluate AFB1, both before and after applying the steam decontamination procedure to detoxify aflatoxin contamination. Cultural assay was used to find the source of aflatoxin. PDA and SDA agar were used to detect aflatoxin producing fungal species. Lactophenol Cotton Blue (LCB) staining was used for microscopic identification of fungal contaminants. Paddies from most regions appeared yellowish in color and odorless with the exception of Hafizabad, Jhang, Sheikhupura, and Gujarat (in Punjab), where they appeared blackish yellow in color and with a pungent smell. According to the results, 43 (61.42%) samples were detected with aflatoxin B1 out of 70 samples, with an average of 15.86±1.7 ug/kg, before treating with seam. After treatment with steam, 7 (10%) samples were detected with aflatoxin B1, with an average of 2.55±1.51 ug/kg. Most regions showed the presence of P. chrysogenum with the exception of R.Y. Khan, which showed the presence of A. niger. Steam, on average, reduced aflatoxin to 51.42%. The current study indicates that steam is an effective treatment to eradicate aflatoxin at industrial level. New approaches may be explored to target the contaminants in order to ensure food safety.

Prevalence of brucellosis among patients attending Wau Hospital, South Sudan.

Brucellosis is a zoonotic disease of public health importance; its prevalence varies globally. In low-income countries, brucellosis is an endemic and neglected disease affecting both animals and humans. This study was intended to establish brucellosis sero-prevalence among patients attending Wau hospital, South Sudan. Across sectional study, was done among randomly selected patients attending Wau hospital. Data was collected using questionnaires and laboratory investigations. Rose Bengal plate Test (RBPT), Serum agglutination test (SAT) and Competitive Enzyme Linked Immuno Sorbent Assay (c-ELISA) was used in the analysis of blood samples serially starting with RBPT which is more sensitive and least specific then SAT. c-ELISA test which is most specific and less sensitive compared to RBPT and SAT was then used to confirm presence of Brucella antibodies in the samples. A total of 416 participants out of 1664 were enrolled to this study. The majority of participants were between 7-to-76 years of age with mean age of 30.72 (SD+/- 12.83). The sero-positivity of patient’s blood samples for brucellosis using c-ELISA was 23.3% (97/416) among patients presenting to Wau hospital. Socio-demographic characteristics, occupation, clinical signs of disease and types of animals reared by animal owners showed no significant correlation with occurrence of sero-positivity among patient’s blood samples for brucellosis. While ethnicity (Nilotic), knowledge of zoonotic disease, and consumption of animal urine were statistically significant (p<0.05). The study found a high prevalence of brucellosis among febrile patients attending Wau hospital general outpatient clinic. There is need for co-ordination and collaboration between veterinary and health sectors of government to help prevent and control brucellosis in the region.

The sero-prevalence of brucellosis in cattle and their herders in Bahr el Ghazal region, South Sudan.

BackgroundBrucellosis is a worldwide recognized bacterial zoonotic disease. There is currently no information on bovine brucellosis sero-prevalence in South Sudan regardless of the economic, social and public health impact on populations. Therefore, for the first time in 33 years, we report the sero-prevalence of brucellosis in cattle and their herders. Furthermore, we characterize the drivers associated with the disease at the human-animal interface in Bahr el Ghazal region, South Sudan.MethodsA total of 893 and 87 animal and human sera respectively were examined between December 2015 and May 2016. Rose Bengal Plate Test (RBPT) and Competitive Enzyme Linked Immuno Sorbent Assay (c-ELISA) were used in parallel to detect anti-Brucella antibodies. Questionnaires were administered to collect relevant metadata used for the association analysis in R version 3.2.3. Odds Ratio (OR) and Confidence Intervals (CI) were determined.ResultsOverall bovine brucellosis prevalence was 31% (95%CI = 28.0–34.2), with the highest 63% (95%CI = 53–70) and lowest 10% (95%CI = 4.5–20.1) prevalence estimates in Wau and Gogrial states respectively. The bovine sero-prevalence was approximately equally distributed among the male 30.4% (26.9–34.2) and the females 32.5% (26.8–38.7). Poor body condition (OR = 0.22; 95%CI = 0.07–0.54) and larger herd sizes (OR = 0.05; 95%CI = 0.008–0.173) were protective factors for brucellosis, while the opposite was true for the second (OR = 1.70; 95%CI = 1.08–2.67) and third (OR = 2.5; 95%CI = 1.46–4.47) lactation stage. The overall brucellosis sero-prevalence in herders was estimated at 33.3% (23.9–44.3).ConclusionWe report a high prevalence of anti-Brucella antibodies in cattle and their herders in Bahr el Ghazal, indicating an enzootic status in the cattle population being an important source of infection for humans. This represents a genuine public health challenge. Therefore, there is need to raise awareness and build capacity and infrastructure in this fragile state to underwrite future public health strategies for brucellosis.

English

A heterologous indirect competitive enzyme linked immuno sorbent assay (icELISA) has been developed for the determination of enrofloxacin (ENR) residues in poultry. For this purpose, carbodiimide active ester method was employed to synthesize the artificial antigen of ENR- bovine serum albumin (BSA) while mixed-anhydride technique was used to synthesize the coating antigen of ENR- ovalbumin (OVA), to pursue the heterologous sensitivity. By square matrix titration, an icELISA method was developed, and the linear range was from 0.02 to 86.3 ng/mL, with limit of detection (LOD) and IC50 value of 0.8 and 0.01 ng/mL, respectively. After optimization, 5% of NaOH was used in the assay buffer and this ELISA system can tolerate methanol not higher than 30%. The correlation coefficients (R2) between concentration spiked and concentration determined were 0.9975 in chicken muscle and 0.9959 in duck muscle, respectively. Therefore, this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of ENR residue in poultry muscles. Key words: Enrofloxacin, artificial antigen, polyclonal antibody, indirect competitive enzyme linked immuno sorbent assay (ELISA), heterologous, poultry.

Seroepidemiology of bluetongue disease in small ruminants of north-east of Iran

ObjectiveTo estimate the prevalence and distribution of bluetongue virus antibody in sheep and goats in 25 townships of Khorasan Razavi. Bluetongue is an infectious, non-contagious, arthropod born viral disease of ruminants and has been reported from most of the tropical and subtropical regions of the world. MethodsA total number of 1 034 serum samples from sheep and goats were collected and transmitted to Serological Laboratory of Veterinary Council of Khorasan Razavi. Serums were screened for the presence of group-specific bluetongue virus antibody using competitive Enzyme Linked Immuno Sorbent Assay (c-ELISA). ResultsThe seropositivity of sheep and goats for bluetongue was found to be 89.2%. The highest prevalence rate was seen in Taybad, Khalil-abad and Torbat-jam (100%) and the least prevalence rate was seen in Jovein (55%). ConclusionsThe results showed that the majority of animals in the north-east of Iran are infected with bluetongue virus. High correlation between abortion history and seroposivity emphasize the economical importance of bluetongue virus in the sheep herds of the region.

Occurrence of mycotoxins in feed samples in Burdur Province, Turkey

The aim of this study was to evaluate the incidence of total aflatoxin (AF), ochratoxin A, T-2 toxin, deoxynivalenol (DON), zearalenone (ZEA), and fumonisin (FB) in dairy cattle, beef cattle, and lamb-calf feeds. A total of 180 dairy cattle, beef cattle, and lamb-calf feeds (60 samples each) were randomly collected from farms, feed mills, and villages in Burdur province, between September 2006 and August 2007. All samples were analyzed by the competitive Enzyme Linked Immuno Sorbent Assay (ELISA). The most frequent mycotoxin detected was total AF, which was found in 108 samples (60 %) in concentrations ranging from 3.82 to 116.83 μg kg(-1), followed by DON that was detected in 87 samples (48.3 %), in concentrations ranging from 18.50 to 500 μg kg(-1). Ochratoxin A (OTA), T-2 toxin, ZEA, and FB were found in 84 (46.7 %), 85 (47.2 %), 57 (31.7 %), and 19 (10.6 %) samples, respectively, in concentrations of 1.01 to 15.85 μg kg(-1) for OTA, 3.85 to 52.36 μg kg(-1) for T-2 toxin, 2.10 to 29.30 μg kg(-1) for ZEA, and 2.69 to 4.96 mg kg(-1) for FB. It was concluded that feed samples in Burdur province were contaminated by mycotoxins, and the levels of total aflatoxin in the samples were considered a risk to animal health.

Serological survey of cattle brucellosis in Eldein, eastern Darfur, Sudan

The study was carried out to determine the prevalence of the brucellosis in cattle in Eldein area, Eastern Darfur state, Western Sudan. Rose Bengal Plate Test (RBPT), Serum Agglutination Test (SAT) and Competitive Enzyme Linked Immuno Sorbent Assay (cELISA) were used for the diagnosis of the disease. RBPT showed 21 (8.4%) positive results, SAT showed 50(20%) positive sample and cELISA showed 5(2%) positive samples. Twenty four (9.6%) cows had a history of abortion, 20 (8%) had histories of retained placentas and 3 (1.2%) had knee hygromas. The study revealed that sex, age and breed were not associated with the brucella seropositivity (P>0.05). In this study, three types of serological test were used, namely RBPT, SATand cELISA and there was significant difference between any two given tests (P<0.05). Using agreement between tests (Kappa Statistic) indicated that RBPT and SAT had moderate agreement (Kappa=0.547), RBPT and cELISA had fair agreement (Kappa=0.364) and SAT and cELISA had slight agreement (Kappa=0.158). cELISA is the most sensitive and reliable test in field. Key words: Brucellosis, cattle, serological tests, prevalence, Sudan.

Sequential injection-ELISA based system for online determination of hyaluronan

Sequential injection-bead-based immunoassay system has been developed. The main purpose is to make immunoassay process more automated by manipulating the precise delivery of micro-volumes of reagents and the precise timing of incubation and washing steps with a computer program that controls the bi-directional syringe pump. The manifold was designed with the aims of reducing back pressure from beads that act as solid surfaces for immobilization of the target substance, reducing dispersion and dilution of the reagent during incubation, and maximizing signal while minimizing incubation time. This was done by introducing air segment to separate the reagent zone from the carrier stream and by using a suitable sensitive detector which, in this case, was an amperometer. In this study, hyaluronan (HA) was used as a target analyte because of its clinical significance as a potential biomarker for liver, bone and cancer diseases. Amount of hyaluronan was determined using competitive enzyme linked immuno sorbent assay (ELISA) based technique where immobilized HA and HA in solution compete to bind with a fixed amount of biotinylated HA-binding proteins (b-HABPs). Upon separation of the two phases, anti-biotin conjugated with enzyme and a suitable substrate were introduced to follow the binding reaction of the immobilized HA and b-HABPs whose degree of binding is indirectly proportional to the amount of HA in solution. A calibration curve was constructed from a series of concentrations of HA standards. Lowest detectable concentration was found to be 1 ng/mL with the dynamic working range of 1–5000 ng/mL and R.S.D. of intra-assay ( n = 7) and inter-assay ( n = 3) of various HA concentrations were 4–10% and 9–12%, respectively. Used beads could be reused by washing with 2 M guanidine. Total analysis time for this automatic assay was about 30 min as compared to the 5–8 h used in conventional batch well ELISA. The system could be applied to assay HA in human serum.

Development of a competitive enzyme linked immuno sorbent assay to measure ceruloplasmin in gamma-irradiated rat serum.

The concentrations of ceruloplasmin could not be determined by comparing the oxidase activities toward p-phenylenediamine and o-dianisidine in rat whole serum with those of purified ceruloplasmin because of the different specific amine oxidase activities of ceruloplasmin purified from normal and gamma-irradiated rats and the lability caused by freezing and thawing. A competitive enzyme linked immunosorbent assay with antigens immobilized on the solid phase was developed to measure the ceruloplasmin in rat serum. The blocking materials and pH of the coating buffer had an effect on the amount of ceruloplasmin bound to the microtiter plates. The blocking materials nonspecifically reacted with the rabbit anti-rat ceruloplasmin IgG, and consequently the coating sensitivity was decreased and standard curves were not formed completely. Because rabbit anti-rat ceruloplasmin IgG did not show any cross reactivity with rat albumin and hemoglobin, these proteins did not affect the assay at a concentration of 200 micrograms/ml. This assay is useful for measuring the concentration of ceruloplasmin in normal and irradiated rat serum.

Development of a competitive enzyme linked immunosorbent assay to measure alpha 2-macroglobulin in irradiated rat serum.

A competitive enzyme linked immunosorbent assay with antigen immobilized on the solid phase was developed to measure alpha 2-macroglobulin in rat serum. The cross reactivity with albumin and hemoglobin was eliminated by use of IgG fractions that were isolated after chromatography on Cibacron Blue F3-GA Sepharose immobilized rat whole serum and hemoglobin Sepharose. Blocking materials and pH of the coating buffer had no effect on the amount of alpha 2-macroglobulin that binds to the plate. When the coating amount of alpha 2-macroglobulin was 100 ng/well, at pH 7.4, 10 mM Tris-HCl containing 150 mM sodium chloride and the IgG amount added was 60 ng/well, then albumin and hemoglobin did not affect the assay at concentrations of 150 micrograms/ml or 200 micrograms/ml. This assay is useful for measuring the concentration of alpha 2-macroglobulin in normal and irradiated rat serum.