Commercial ELISA Research Articles

Electrochemical immunosensing of Crohn's disease biomarkers using diazonium salt-grafted crystalline nanocellulose/carbon nanotube-modified electrodes.

Thefirst dual immunosensoris reportedfor the determination of IL-12 and IL-23, two relevant biomarkers of Crohn's disease (CD). The strategy relies on the selective capture of the targets by the respective antibodies which were covalently immobilized onto SPCEs modified with crystalline nanocellulose (CNC) and multi-walled carbon nanotubes (MWCNTs) followed by conjugation with a detector antibody labelled with poly-HRP-Strept and amperometric transduction using the H2O2/HQ system. The combination of CNC, a nanomaterial scarcely exploited in immunosensing, with MWCNTs enables the preparation of a novel dual immunosensor for the determination of CD biomarkers in clinical samples, including faeces. The developed bioplatform exhibits a high reproducibility and selectivity allowing the simultaneous determination of IL-12 and IL-23 using a smaller sample volume and in a much shorter assay time than those reported for each target with commercial ELISA kits. The dual immunosensor was successfully applied to the analysis of raw serum and faeces spiked with IL-12 and IL-23 at the expected levels in patients with severe CD.

Electrochemical immunosensor for the predictive cancer biomarker SLFN11 using reduced graphene oxide/MIL-101(Cr)-NH2 composite

SLFN11 is a predictive cancer biomarker essential for identifying tumors that are sensitive to DNA-damaging agents, facilitating more personalized and effective treatment approaches. Detecting this biomarker can guide therapeutic decisions and improve outcomes for cancer patients. However, existing detection methods for SLFN11 are complex and require advanced techniques. In this study, we introduce the first immunosensor designed for on-site detection of SLFN11. An advanced electrochemical immunosensor platform utilizing a composite of graphene oxide (GO) and chromium-based metal organic framework (MIL-101 (Cr)-NH2) was developed. The integration of GO and MIL-101(Cr)-NH2 was characterized through FT-IR, XRD, SEM, and XPS, affirming the formation of the composite. The subsequent electrochemical reduction to rGO/MIL-101(Cr)-NH2 significantly improved the electrochemical performance and stability. A glutaraldehyde cross-linker was then utilized to attach the SLFN11-specific antibody to the amine groups of the MOF-modified electrodes. This led to the development of rapid, sensitive, portable, and cost-effective immunosensor for SLFN11 at concentrations as low as 8.9 pg/mL which holds promise for early cancer diagnosis. High specificity was achieved, with minimal cross-reactivity observed with other cancer biomarkers such as pepsinogen I, claudin 18.2 and Programmed cell death protein 1. Demonstrating practical applicability, the electrochemical immunosensor validated by commercial ELISA kit showed successful detection in serum samples with high recovery rates and reproducibility. This research highlights the potential of rGO/MOFs composites in electrochemical biosensors developments for early cancer diagnostics and personalized medicine.

Comparative assessment of commercial ELISA kits for the screening of chloramphenicol residues in meat and aquaculture products according to European Regulation (EU) 2021/808 and to the new Reference Point for Action (Commission Regulation (EU) 2019/1871)

This study covered the evaluation of performance characteristics and validation of five commercial ELISA kits for the detection of a banned antimicrobial, chloramphenicol (CAP), in muscle and aquaculture products. CAP has been banned in the European Union since 1994, but is still authorised in some countries in the world. In 2019, the European Union set a new Reference Point for Action (RPA), decreasing the acceptable limit of CAP in animal tissues for human consumption from 0.30 µg/kg to 0.15 µg/kg. Validations were performed according to the European Regulation EC/2021/808 and to the European Guideline on Screening Method Validation (2023). The detection capabilities CCβ were all determined under the RPA, but were 3 to 15 times higher than the announced limit of detection (LOD). False negative rates were satisfactory for all the kits (≤ 5%) and false positive rates were acceptable. All of them were found out to be applicable to aquaculture products and meat at a common CCβ.

Longitudinal serological and virological survey of hepatitis E virus in wild boar (Sus scrofa majori, Maremman wild boar) and fallow deer (Dama dama) populations in a protected area of Central Italy

Hepatitis E virus (HEV) is recognized as an emerging zoonosis. Pigs and wild boars are considered the main reservoirs of zoonotic HEV-3 and HEV-4 genotypes. In Europe, autochthonous human cases of hepatitis E, mainly associated with HEV-3 and consumption of raw or undercooked pig and wild boar liver/meat, have increased over the last decades. From 2016 to 2024, during several hunting seasons, we conducted a molecular and serological longitudinal survey on the circulation of HEV in Maremman wild boar (Italian subspecies/ecotype, Sus scrofa majori) and fallow deer (Dama dama) populations in a protected area in Central Italy. During the study period, 346 livers (256 from wild boar, 90 from fallow deer), 161 serum (127 from wild boar, 34 from fallow deer), and 23 meat juice (11 from wild boar, 12 from fallow deer) samples were collected. Serum and meat juice samples were tested using a commercial ELISA test for the detection of total anti-HEV antibodies. An estimated serological prevalence of 28.3% (39/138) in wild boar and 21.7% (10/46) in fallow deer was found. The 346 liver samples were tested using a HEV Real-Time RT-PCR for the detection of HEV-RNA. Thirty-one wild boar (12%) and four fallow deer (4.4%) livers were found positive. Phylogenetic analysis of 11 partial ORF2 sequences from wild boar confirmed the HEV3 heterogeneity in this species, revealing different strains (3f, 3c) circulating over the years. The detected subtypes are among the most commonly detected in Italy and our strains showed a high correlation with human and wild boar Italian strains. Although the studied area is a fenced natural reserve, the presence of different strains over time suggests the probable virus introduction from the external. Our results confirm fallow deer susceptibility to the infection, and that wild boar could be considered the main wild HEV reservoir. This is also the first study demonstrating the infection in the so-called Italian subspecies/ecotype Maremman wild boar. Moreover, our results corroborate that the consumption of undercooked or raw liver from both wild boar and fallow deer, or the direct contact with these animals, could represent a zoonotic risk.

Quantum dot-based biomimetic fluorescence immunoassays for enrofloxacin detection in animal-derived foods.

Long-term consumption of foods with excessive enrofloxacin (ENRO) residues may cause the accumulation of ENRO in the human body, thus damaging human health. In this study, quantum dot-based biomimetic fluorescence immunoassays were used for enrofloxacin detection in food of animal origin. Under the most suitable conditions, the detection limit (IC15) of the method in standard solution was 0.13 ± 0.02 ng L-1 and the sensitivity (IC50) was 0.13 ± 0.18 μg L-1. The recoveries for ENRO from four fortified food samples, including chicken, eggs, shrimp, and milk, ranged from 85.74% to 114.19% and the coefficients of variation were 0.01-18.09%. The established method shows good agreement with the results obtained using commercial ELISA kits, with a correlation coefficient of 0.997. The proposed method shows the advantages of high sensitivity, specificity and wide detection range. It can be used as an alternative method for the rapid and sensitive detection of ENRO in food of animal origin.

Seroprevalence of Mycobacterium avium subsp. paratuberculosis Infections in Small Ruminants in Europe

Paratuberculosis, also known as Johne’s disease, is a granulomatous enteritis in both domestic and wild ruminants caused by the bacterium Mycobacterium avium subsp. paratuberculosis. Understanding the prevalence of this disease in small ruminants is essential for disease control and prevention strategies. A systematic review of the literature was conducted using the PubMed, ScienceDirect and Scopus databases to identify all articles reporting Mycobacterium avium subsp. paratuberculosis (MAP) seroprevalence in sheep and goats in Europe, published from January 2006 to December 2023. The initial search for existing publications reporting systematic reviews and primary studies was carried out by searching the available databases. For the final selection of studies, an initial screen for basic eligibility and a detailed appraisal of quality were performed. After the study selection, the relevant data was extracted. The detailed appraisal generated 21 publications that reported 55 studies, 22 (40.0%) from sheep (12 at the animal-level and 10 at the flock-level) and 28 (50.9%) from goats (17 at the animal-level and 11 at the flock-level), and 5 (9.1%) from mixed small ruminant species at the animal level. In total, 34 (61.8%) were animal-level studies and 21 (38.2%) were flock-level studies. Population and inclusion criteria were highly variable among studies. Sample sizes ranged from 291 to 15,585 animals. Most studies reported testing adult animals (over 24 months of age). Commercial ELISA kits were used in most studies. The highest prevalence was obtained in sheep (100%) in Türkiye, and the lowest was found also in sheep (0.7%) in Austria. Overall, the results suggest that MAP antibodies have been frequently detected among small ruminants in some countries and there is a need for standardisation of case definitions to improve the accuracy of prevalence estimates. Further research is needed to understand the risk factors associated with MAP infection in small ruminants and to develop effective control and prevention strategies.

Seroprevalence and associated risk factors for Fasciola hepatica in sheep in Nile Delta of Egypt

Fascioliasis is a globally distributed zoonotic parasitic disease that affects ruminants, including sheep. This study conducted from January to December 2023, aimed to determine the prevalence of Fasciola hepatica in sheep across three governorates in Egypt's Nile Delta and to assess associated risk factors. A total of 455 serum samples were analyzed using a commercial ELISA kit, revealing antibodies against F. hepatica in 22.2 % of the tested sheep. There was no significant association between locality or sex and the seroprevalence of F. hepatica in sheep; however, the highest prevalence was observed in Kafr ElSheikh and in female sheep.Concerning risk factors, poor conditioned sheep aged between 1 and 2 years showed 2.1 and 3.8 times higher of infection probability than others. In addition, the risk of F. hepatica infection in sheep increased significantly in winter season (OR = 6.6, 95 %CI: 2.6–16.8), in absence of prophylactic treatment (OR = 2.2, 95 %CI: 1.3–3.3) and in presence of snail (OR = 3, 95 %CI: 3–5.4). The existence of antibodies against F. hepatica in examined sheep raising in Nile Delta indicating that the disease is reported in the studied areas and needs to be managed on farms through control and preventative measures.

Comparison of the Diagnostic Performance of Antigen B Purified from Sheep Hydatid Cyst Fluid (HCF) with Commercial ELISA Kit.

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the metacestode of Echinococcus granulosus. CE is a health problem in Middle Eastern countries, such as Iran. The purpose of this study was to purify subunit 8 KDa antigen B from crude sheep hydatid cyst fluid (HCF) and compare its sensitivity and specificity with a commercial human ELISA kit (PT-Hydatid-96). 28 sera samples were collected from hydatid cyst patients who had surgery for a hydatid cyst and had their disease confirmed by pathology after the surgery. Furthermore, 35 samples of healthy individuals with no history of hydatid cysts were collected, as were nine serum samples from parasite-infected non-CE patients. HCF was obtained from sheep fertile cysts at a Sari slaughterhouse and used as an antigen. In an indirect ELISA test, the B antigen was employed, and the results were compared to those from a commercial ELISA kit. The results of this study were analyzed using the Kappa test. The commercial ELISA kit showed 17 cases (23.6%) positive, 44 cases (61.1%) negative, and 11 cases (15.3%) borderline. B antigen showed that 18 (25%), 43 (59.7 %), and 11 (15.3%) were positive, negative, and borderline, respectively. One sample (1.4% of 72 total samples) of 35 serum samples from healthy individuals was positive using B antigen-based ELISA. In addition, all nine serum samples from parasite-infected non-CE patients were negative for both tests. The sensitivity and specificity of the commercial ELISA kit have been evaluated at 60.7% and 100%, respectively. For B antigenbased ELISA, these values are 64.3 and 97.7%, respectively. Antigen B produced from hydatid cyst fluid is a promising option for serological identification of hydatid cysts in both infected and healthy individuals. In an indirect ELISA test, hydatid fluid antigen could be used as a precise source of detection.

Seroprevalence of Toxoplasma gondii in Cattle in Southern Egypt: Do Milk and Serum Samples Tell the Same Story?

Toxoplasma gondii is an intracellular protozoan parasite of veterinary and public health importance. Infection may lead to abortion in susceptible pregnant animals and women, and potentially fatal health complications in immunocompromised individuals. In this study, we aimed to provide an update on the seroprevalence of, and risk factors for, T. gondii antibodies in cattle from Qena, southern Egypt. Additionally, we investigated if raw milk and serum samples from the same animals reacted similarly in a commercial ELISA, thus potentially reducing the invasiveness of future serosurveillance studies. Cattle serum samples (n = 362) from three locations in the Qena governorate (Qena, Qus, and Al Waqf cities), of both sexes and different ages were collected. From most dairy cows, a corresponding milk sample (n = 154) was additionally obtained. We found that the overall seroprevalence in serum samples was 9.1% (33/362). Increasing age was the sole risk factor identified in our study among all tested parameters (location, age, sex, lactating yes or no). Thus, older cattle (more than 3 years old) exhibited significantly higher rates of T. gondii antibodies (11.7%; p = 0.033, odd ratio = 4.3) in comparison to animals younger than 1 year (2.9%). In the corresponding serum and milk samples, the prevalence was 12.3% (19/154) in serum samples, and 9.7% (15/154) in milk samples, respectively. A high correlation was observed between the two sample types with a concordance of 97.4%, a kappa value of 0.87, and a Pearson r correlation coefficient of 0.85. When the serum ELISA was taken as the gold standard, the milk ELISA had the following characteristics: sensitivity (78.9%), specificity (100%), positive predictive value (100%), negative predictive value (97.1%), and area under the receiver operating characteristic (ROC) curve (0.6, p = 0.0011). In this study, we confirmed the frequent occurrence of T. gondii antibodies in cattle in southern Egypt and demonstrated that non-invasive milk samples may be used instead of serum samples for seroprevalence studies in dairy cows.

Comparison of Three Different IBD Vaccination Protocols in Broiler Chicken-Efficacy, Serological Baselines and Histo-Pathological Lesions in the Bursa of Fabricius.

Infectious bursal disease (IBD) is a highly infectious disease of chicken found in all latitudes, due to the very high resistance to environmental conditions and commonly used disinfectants of the IBD virus (IBDV). This study was conducted to evaluate three IBD vaccination protocols in broiler chicken in terms of their effectiveness (clinical observations and production performance of the flock), estimation of serological baseline values (with the use of two different commercial ELISA kits) and the degree of progression of BF lesions (histopathological lesion score (HLS)) after vaccination. The three protocols were (I) single vaccination using an intermediate plus vaccine, (II) double vaccination with an intermediate vaccine and (III) double vaccination with an intermediate plus vaccine. Birds on farms vaccinated with protocol II were characterized by the lowest antibody titers in both ELISA tests and the lowest average HLS. The highest IDEXX titers were obtained in birds vaccinated with protocol III, while in the BIOCHEK test the highest titers were obtained for birds vaccinated with protocol I. Protocols I and III were characterized by similar HLSs. Birds vaccinated with protocols I and III had higher immune uniformity. The estimated serological baseline values and the degree of HLSs presents a clear picture of the differences between the different vaccination protocols and allows their adaptation for different farms depending on the current IBD epidemiological situation.

Recombinant MS087-based indirect ELISA for the diagnosis of Mycoplasma synoviae.

Accurate detection is a prerequisite for effective prevention and control of Mycoplasma synoviae infection. ELISA is the most popular method for the clinical detection of M. synoviae because of its convenience, low cost, and high detection rate. However, the cross-reactivity of commercially available ELISA kits with other avian pathogen-positive sera needs to be addressed. The aim of this study was to establish an ELISA method with high specificity for the detection of anti-M. synoviae antibodies in chicken serum to evaluate the M. synoviae infection status on poultry farms. The recombinant MS087 (rMS087) protein was expressed in Escherichia coli BL21 (DE3) and purified by Ni2+ affinity chromatography. An antibody against rMS087 was generated by immunizing BALB/c mice. Bioinformatic analysis revealed that MS087 was conserved among M. synoviae strains. Western blotting and indirect immunofluorescence results indicated that MS087 was not only localized in the cytoplasm and on the membrane but also secreted by the organism. For the established ELISA method based on rMS087, the optimal antigen concentration, blocking buffer, blocking duration, serum dilution, serum incubation duration, secondary antibody dilution, secondary antibody incubation duration and colorimetric reaction duration were 2 μg/mL, 1% BSA, 3 h, 1:500, 1.5 h, 1:20,000, 2 h and 5 min, respectively. Validation of the rMS087-based ELISA revealed a cut-off value of 0.5. The coefficients of variation of both the intra-batch and inter-batch methods were less than 9%. The assay was able to differentiate positive serum against M. synoviae from antisera against nine other avian pathogens and was able to recognize M. synoviae-positive sera at a dilution of 1:1,000. Compared with the commercial ELISA method, the rMS087-based ELISA has the potential to recognize more positive sera against M. synoviae. Collectively, the rMS087-based ELISA is a reproducible, specific, and sensitive serological method for detecting antibodies against M. synoviae in chicken serum and has robust potential for large-scale serological epidemiology of M. synoviae infection on poultry farms.

Circulating levels of neutrophil extracellular traps reflects local neutrophil activity but not fibrinolytic activity in coronary thrombi from patients with ST-elevation myocardial infarction

Abstract Introduction Neutrophil extracellular traps (NETs) are associated with impaired fibrinolysis in ischemic stroke, while NETs degradation enhances lysis of coronary thrombi in ex vivo experiments. Whether circulating NET markers are associated with local fibrinolytic activity in patients with ST-elevation myocardial infarction (STEMI) has not been reported. Purpose We investigated associations between circulating NET markers and gene expression of fibrinolytic markers in aspirated coronary thrombi from patients with STEMI. Furthermore, we explored whether local upregulation of NET formation by peptidylarginine deiminase 4 (PAD4) was reflected in the circulation. Methods Coronary thrombi from 35 STEMI patients undergoing primary percutaneous coronary intervention (PCI) were aspirated and promptly snap-frozen to -80°C. Peripheral blood samples were collected simultaneously. Median time from start of symptoms to PCI was 152 min. Thrombus gene expression of tissue Plasminogen Activator (tPA), urinary-type Plasminogen Activator (uPA), plasminogen-activation inhibitor type 1 (PAI-1) and PAD4 were quantified with RT-PCR. Gene expression of PAD4 was also measured in circulating leukocytes. Circulating NET markers were measured by a fluorescent nucleic acid stain using fluorometry (dsDNA), an in-house ELISA technique (MPO-DNA) and commercial ELISA (H3Cit). Correlations were tested using Spearman’s rho. Results There were no correlations between any of the circulating NET markers and gene expression of the fibrinolytic markers tPA, uPA and PAI-1 in the thrombus. Of the circulating NET markers, dsDNA correlated with thrombus gene expression of PAD4 (rs=0.491 p=0.006), whereas none of the NET markers correlated with gene expression of PAD4 in circulating leukocytes. Conclusion Circulating NET markers were not associated with gene expression of fibrinolytic activity in thrombi from patients with STEMI. Local NET formation in the thrombi, measured as PAD4 expression, was associated with the circulating NET marker dsDNA. These findings suggest that circulating NET markers have limited utility for assessing local fibrinolytic activity, but might reflect PAD4-dependent NET formation in coronary thrombi.

Evaluation of Label Compliance to Allergens in Imported Asian Food Sold in Northern Italy

Abstract Background The import of Asian food is strongly increasing in the EU. In Italy, the Asian market has expanded over the last 3 decades. Unfortunately, the labelling of these products often implies errors, mainly due to linguistic differences. Consequently, it can become a consistent problem regarding food safety, in particular for allergens declaration. According to EU Reg. 1169/2011 (Annex II), allergens must be labelled in bold and translated in the current language of the Member State. Asian food non-compliant labelling could enhance the probability of allergic consumers to get adverse events; accordingly, it is important to increase controls on the imported products. The present work investigates the label compliance to allergens on Asiatic products imported in Northern Italy. Methods Totally, 39 foodstuffs were tested for gluten, 93 for white egg proteins and 118 for milk proteins by commercial ELISA kits according to manufacturers’ instructions. Samples were considered non-compliant when the results were above 20 mg/Kg, the law limit for gluten, or above the milk LOD (10 µg/Kg) and the egg LOD (0.4 mg/Kg). Results Data showed 18% (CI 95% 0.09-0.32) of non-compliant samples for gluten: 6 samples > 80 mg/Kg (snacks and ready to eat meal) and 1 sample containing 48 mg/Kg (snack). In addition, 19.4 % (CI 95% 0.13-0.28) showed presence of egg proteins (noodles, snacks, patties and pasty). Finally, 13.6% (CI 95% 0.08-0.21) samples were irregular for undeclared milk (noodles and snacks). Conclusions Products label report their content and represent an important element for food trading and tracing. The results obtained confirm a high percentage of non-compliant labels, underlining how the consumption of imported products can represent a health risk for allergic consumers. The present work highlights the importance of imported food monitoring to guarantee the consumers health, and of the implementation of standardized/international regulated labelling rules. Key messages • Many non-compliant labels about allergens was observed on Asiatic products. • Correct labelling of allergens in imported food should be monitored to avoid adverse events.

Canine leishmaniasis caused by Leishmania tropica in southeastern Iran: a case series study

Dogs are the primary reservoirs of Leishmania infantum (L. infantum), but Leishmania tropica (L. tropica) infection is also possible in dogs and can transmitted to humans. The southeast of Iran experiences a high prevalence of canine leishmaniasis (CanL), and veterinarians frequently encounter symptomatic cases. Therefore, from December 2017 to November 2022, the present case series was designed to assess the prevalence of CanL in owned dogs with cutaneous manifestations resembling CanL. A total of 500 owned dogs with dermal lesions from two endemic cities, Zabol and Kerman, were enrolled. Impression smears from skin lesions and popliteal lymph nodes were prepared from all cases, whereas blood samples were gathered from amastigote-positive dogs for serological and molecular surveys. Commercial ELISA was done on sera samples, and two-step nested PCR was used on extracted DNA to amplify variable fragments of the Leishmania species’ kinetoplast DNA (kDNA). Leishman bodies were microscopically detected in 7.2% (36/500) of dermal smears. Of the 360 owned dogs from Zabol, 2 have been diagnosed with L. tropica, and 10 have been diagnosed with L. infantum. Out of 140 owned dogs from Kerman, 8 were identified with L. tropica infection, and 16 were infected with L. infantum. Molecular results revealed the presence of 750 full dual-band bases related to the L. tropica species in 10/36 (27.7%) cases, which showed a considerable increase in canine cutaneous leishmaniosis compared with previous studies in southeastern Iran. The noticeable prevalence of L. tropica in owned dogs indicated that the dog’s role in cutaneous leishmaniosis should be re-evaluated as a possible animal reservoir in endemic areas.

Seroprevalence of antibodies against diphtheria, tetanus, and pertussis across various age groups during the post-COVID-19 pandemic period in Chonburi Province, Thailand

BackgroundLimited data exists regarding population immunity against diphtheria, tetanus, and pertussis in Thailand during the post-COVID-19 pandemic period. This study aimed to evaluate the age-specific seroprevalence of anti-diphtheria toxoid (anti-DT) IgG, anti-tetanus toxoid (anti-TT) IgG, and anti-pertussis toxin (anti-PT) IgG in individuals across diverse age groups in Chonburi province, Thailand following the COVID-19 pandemic. MethodsBetween October 2022 and January 2023, a total of 657 participants from Chonburi Province, Thailand, were included in this study. The participants were categorized into 9 age groups: <5, 5–10, 11–20, 21–30, 31–40, 41–50, 51–60, 61–70, and >70 years. Analysis of anti-DT, anti-TT, and anti-PT IgG levels was conducted using commercial ELISA kits (EUROIMMUN, Lübeck, Germany). ResultsOverall, 65.4 % of the population had seroprotection against diphtheria (antibody level ≥0.1 IU/mL), while 95.1 % had seroprotection against tetanus (antibody level ≥0.1 IU/mL). The 31–40 years age group exhibited the lowest seroprotection for diphtheria (48.9 %), and the >70 years age group had the lowest seroprotection for tetanus (73.3 %). The <5 years age group showed the highest seropositive rate and highest geometric mean titers for anti-PT IgG. On the contrary, the majority of individuals over 11 years of age displayed anti-PT IgG levels below 40 IU/mL. ConclusionsTo prevent diphtheria and pertussis outbreaks in Chonburi province, implementing catch-up vaccination is necessary. Targeted interventions should be deployed to enhance vaccination coverage among the susceptible population.

Seroepidemiology of Coxiella burnetii in Domestic and Wild Ruminant Species in Southern Spain.

The European Food Safety Authority has recently listed Q fever as a priority for setting up a coordinated surveillance system. Although Spain is the country with the highest human incidence of the disease in the European Union, updated data on Coxiella burnetii in ruminants are still limited. A total of 780 serum samples from small ruminants and 605 sera from wild ruminants were collected in the Mediterranean ecosystems of southern Spain during the period 2015-2023. Anti-C. burnetii antibodies were detected using a commercial indirect ELISA. The overall individual seroprevalence in the small ruminants was 49.1% (383/780; 95% CI: 45.6-52.6). Antibodies against C. burnetii were identified in 40.0% (156/390) of sheep and in 58.2% (227/390) of goats. At least one seropositive animal was observed in all sheep (100%) and in 92.3% of goat flocks. The species (goat) and the existence of reproductive disorders in primiparous females were potential risk factors for C. burnetii exposure in small ruminant farms. In the wild ruminants, the overall seroprevalence against C. burnetii was 1.5% (9/605; 95% CI: 0.8-2.8). Anti-C. burnetii antibodies were found in 1.8% (2/110) of mouflon, 1.5% (6/390) of red deer, and 1.0% (1/105) of Iberian ibex. The high exposure of the small ruminants to C. burnetii, particularly in goats, detected in the present study is of animal and public health concern. Our results denote that wild ruminants only play a minor role in the epidemiology of this bacterium in southern Spain and suggest an independent epidemiological cycle of C. burnetii in domestic and wild ruminant species in the study area.

The herd-level prevalence of caprine arthritis-encephalitis and genetic characteristics of small ruminant lentivirus in the Lithuanian goat population

Caprine arthritis-encephalitis (CAE) is a progressive disease of goats caused by small ruminant lentivirus (SRLV) and is considered as one of the most important threats for goat farming in developed countries. The disease prevalence has never been investigated in the Lithuanian goat population. Therefore, a descriptive cross-sectional study was carried out in 2021–2022 to determine if SRLV infection was present in the Lithuanian goat population and, in the case of a positive result, to estimate the true herd-level prevalence of SRLV infection and specify genotypes and subtypes of SRLV responsible for the infection. Thirty goat herds counting >5 adult goats were randomly selected and, in each herd, a representative sample of adult goats was blood-sampled and tested serologically for SRLV infection using a commercial ELISA. The herd was considered infected if at least one goat tested positive and the true herd-level prevalence of SRLV infection was estimated using the Bayesian approach. Seropositive animals were found in 17 / 30 herds (57 %; 95 % confidence interval: 39 %, 73 %). The true herd-level prevalence was 56 % (95 % credible interval: 36 %, 76 %). In 10 / 17 seropositive herds whose owners consented for resampling of seropositive goats, 1–5 seropositive goats were tested using the nested real-time PCR (nRT-PCR). Goats from 9 seropositive herds tested positive in the nRT-PCR: in 4 herds for genotype A, in 4 herds for genotype B, and in 1 herd – 2 goats for genotype B and 1 goat for genotype A. From each of 9 nRT-PCR-positive herds, 1 PCR product of each genotype was sequenced using Sanger method and the phylogenic tree was constructed using the neighbor-joining method in the Molecular Evolutionary Genetics Analysis software. Four herds turned out to be infected with B1 subtype (91 % identity with the prototypic strain), 3 herds with A2 subtype (90 %-92 % identity), and a herd with mixed infection was infected with B1 (91 % identity) and A2 subtype (90 % identity). In one herd, the only seropositive goat was found to be infected with the strain most closely related to the A1 subtype (80 % identity). This study shows for the first time that SRLV infection is present and widespread in the Lithuanian goat population and both classical SRLV genotypes, represented by quite typical subtypes A2 and B1, appear to be responsible for the infection.

Detection of Salmonella by competitive ELISA of lipopolysaccharide secreted into the culture medium

Detection of Salmonella in food is topical due to known cases of salmonellosis epidemics. Immunochemical methods including ELISA are widely used for Salmonella detection. Traditionally, commercial ELISA kits are based on sandwich technique and detect lipopolysaccharide (LPS), which is considered to be the component of the outer membrane of Gram-negative bacteria. Our aim was elaboration of competitive ELISA test for Salmonella detection in food with improved parameters. It was shown that in the Salmonella culture after the standard sample preparation procedure LPS is present mainly outside cells as a component of outer membrane vesicles. Improved sample preparation procedure includes separation of bacteria from the medium and analysis of the medium, which increases analytical sensitivity. Immobilization of the bovine serum albumin (BSA)-LPS conjugate in microplate wells allows to obtain a more homogeneous coating than immobilization of LPS itself. Thus, we have developed test system for Salmonella detection in food by competitive ELISA of LPS secreted into the culture medium with the immobilized BSA-LPS conjugate and monoclonal antibodies (mAb) to LPS core in the liquid phase. New competitive ELISA test is high sensitive, give reproducible results, allows the detection of any Salmonella serotype and is important for the protection of human health.

Transplacental Transfer of Oxytocin and Its Impact on Neonatal Cord Blood and In Vitro Retinal Cell Activity.

The development of fetal organs can be impacted by systemic changes in maternal circulation, with the placenta playing a pivotal role in maintaining pregnancy homeostasis and nutrient exchange. In clinical obstetrics, oxytocin (OXT) is commonly used to induce labor. To explore the potential role of OXT in the placental homeostasis of OXT, we compared OXT levels in neonatal cord blood among neonates (23-42 weeks gestation) whose mothers either received prenatal OXT or experienced spontaneous labor. Our previous research revealed that the oxytocin receptor (OXTR), essential in forming the blood-retina barrier, is expressed in the retinal pigment epithelium (RPE). We hypothesized that perinatal OXT administration might influence the development of the neural retina and its vasculature, offering therapeutic potential for retinal diseases such as retinopathy of prematurity (ROP). Plasma OXT levels were measured using a commercial OXT ELISA kit. Human fetal RPE (hfRPE) cells treated with OXT (10 µM) were assessed for gene expression via RNA sequencing, revealing 14 downregulated and 32 upregulated genes. To validate these differentially expressed genes (DEGs), hfRPE cells were exposed to OXT (0.01, 0.1, 1, or 10 µM) for 12 h, followed by RNA analysis via real-time PCR. Functional, enrichment, and network analyses (Gene Ontology term, FunRich, Cytoscape) were performed to predict the affected pathways. This translational study suggests that OXT likely crosses the placenta, altering fetal OXT concentrations. RNA sequencing identified 46 DEGs involved in vital metabolic and signaling pathways and critical cellular components. Our results indicate that the perinatal administration of OXT may affect neural retina and retinal vessel development, making OXT a potential therapeutic option for developmental eye diseases, including ROP.

A 50-year-old question: Can imidocarb chemoprophylaxis ensure seroconversion for babesiosis in cattle under field conditions?

Bovine babesiosis, caused by Babesia bovis or Babesia bigemina, is a major tick-borne disease affecting livestock. In regions with limited vaccine availability, imidocarb is widely used as a chemoprophylactic drug. Although it is assumed that chemoprophylaxis allows for the development of immunity shortly after treatment, the extent of seroconversion during the imidocarb administration protocol remains largely unexplored, with most investigations emphasizing symptom prevention. This research endeavors to verify the seroconversion rate (humoral immunity) of cattle undergoing imidocarb chemoprophylaxis while exposed to tick vectors in field conditions. Fifteen tick-naïve heifers were used, with twelve receiving imidocarb (experimental group) on day 0 of the experiment, and the remaining three serving as controls. On day one of the study, all animals were introduced into a tick-infested pasture. Subsequently, at 28-day intervals (days 28, 56, 84, 112, 140, and 168), the experimental group received imidocarb treatments (1.2 mg/Kg). The detection of antibodies against B. bovis and B. bigemina was performed using commercial ELISA kits. Throughout the study, all animals were exposed to natural infestation by Rhipicephalus microplus ticks. By the 56th day, after two imidocarb doses, 25 % of the experimental group had seroconverted for B. bovis, and 41 % for B. bigemina. By the 84th day, 66 % were seropositive for B. bovis and B. bigemina. By the 112th day, 75 % were seropositive for B. bovis. Notably, one heifer (8 %) failed to seroconvert for either species, while 41 % remained seropositive for only one Babesia species. These findings underscore certain limitations of the chemoprophylaxis protocol for bovine babesiosis. While the majority of treated cattle become seropositive for at least one Babesia species after four successive treatments, exposure to the parasite while receiving imidocarb chemoprophylaxis does not guarantee seroconversion for all treated animals.