Concordance between two monoclonal antibody-based antigen detection enzyme-linked immunosorbent assays for measuring cysticercal antigen levels in sera from pigs experimentally infected with Taenia solium and Taenia hydatigena.

BACKGROUND: Antigen detection in Taenia solium cysticercosis confirms viable infection in the intermediate host (either pig or human). The reference B158/B60 monoclonal antibody (mAb)-based Ag-enzyme-linked immunosorbent assay (ELISA) has acceptable levels of sensitivity and specificity in human neurocysticercosis with multiple brain cysts, although its sensitivity is lower in cases with single brain cysts, whereas in porcine cysticercosis the assay specificity is affected by its frequent cross-reaction with Taenia hydatigena, another common cestode found in pigs. Our group has produced 21 anti-T. solium mAbs reacting against antigens of the whole cyst, vesicular fluid, and secretory/excretory products, identifying TsW8/TsW5 as the most promising pair of mAbs for an Ag-ELISA. METHODS: We report the use of the TsW8/TsW5 Ag-ELISA to measure cysticercus antigen levels [expressed as optical density (OD) values] in two panels of sera collected from day 0 (baseline) to day 90 postinfection (PI) from pigs experimentally infected with T. solium (n = 26) and T. hydatigena (n = 12). At baseline and on days 28 and 90 PI, we used Bland-Altman (BA) analysis and Lin's concordance correlation coefficients (CCC) to determine the concordance between the TsW8/TsW5 and the B158/B60 Ag-ELISA. RESULTS: The TsW8/TsW5 Ag-ELISA was able to efficiently measure circulating antigen levels in T. solium-infected pigs, similar to that obtained with the B158/B60 Ag-ELISA. Almost all paired log-OD differences between assays were within the limits of agreement (LoA) in the BA analysis at baseline and on days 28 and 90 PI (92.3%, 100%, and 100%, respectively), and a high concordance of log-ODs between assays was also found (Lin's CCC: 0.69, 0.92, and 0.96, respectively, all P < 0.001). In pigs infected with T. hydatigena, almost all paired log-OD differences were within the LoA in the BA analysis, whereas the concordance of log-ODs between assays was low at baseline (Lin's CCC: 0.24) but increased on days 28 and 90 PI (Lins' CCC: 0.88 and 0.98, P < 0.001). CONCLUSIONS/SIGNIFICANCE: The TsW8/TsW5 Ag-ELISA recognizes antigens in pigs with T. solium cysticercosis and is highly concordant with the B158/B60 Ag-ELISA. However, its diagnostic use is hampered by cross-reactions with T. hydatigena, as in other mAb-based Ag-ELISAs.

Evaluation of ELISA in raw milk for detection of antibodies to Mycobacterium avium subsp. paratuberculosis in dairy herd.

Paratuberculosis (PTBC) is a chronic intestinal disease of animals caused by Mycobacterium avium paratuberculosis (MAP). MAP infection is diagnosed through indirect tests based on the immune response. The aims of this study were to compare the performance of two milk ELISA for the diagnosis of PTBC and to assess the bulk tank milk (BTM) MAP exposure in dairy cattle in Argentina. A total of 357 fecal, serum, and milk samples were collected. The fecal samples were processed by culture for MAP isolation, while both, serum and milk samples were used for the detection of antibodies by two different ELISA tests, "in-house" and commercial kit. MAP was isolated in 3.9% of fecal samples. For milk ELISA, poor concordances were obtained. Optimized cut-off points were calculated. The highest sensitivity and specificity values (64% and 80% respectively) were obtained with the combination of MAP isolation and commercial milk ELISA. The results indicate that the combination of different techniques to identify of dairy cattle infected with MAP increases the efficiency of diagnosis. In addition, BTM  samples (n=98) were evaluated to determine herd status using the commercial kit during two seasons, identifying 33.3% of positive samples in autumn and 35.4% in spring.

Comparison of virus-neutralizing and virus-specific ELISA antibody responses among bovine neonates differentially primed and boosted against bovine coronavirus.

Objective: This study addressed the current gap in knowledge of neonatal prime-boost immune responses for the control of bovine coronavirus (BCoV) respiratory disease in weaning-age beef cattle. Animals: Study 1 and Study 2 had 33 and 22 commercial cross neonatal beef calves, respectively. Procedures: Study 1 compared BCoV-neutralizing antibody concentrations of control calves with 3 groups of calves differentially vaccinated with mucosal and/or systemic BCoV modified live virus (MLV) vaccines. Study 2 compared specific and neutralizing antibody concentrations among mucosally BCoV primed groups of calves that were differentially systemically boosted. Results: In Study 1, calves that were mucosally primed and systemically boosted had higher BCoV-neutralizing antibody concentrations than the control group at weaning. In Study 2, boosting mucosally primed calves by injecting inactivated or MLV vaccine resulted in anamnestic BCoV-specific antibody responses at weaning. Conclusion: Neonatal mucosal priming and systemic boosting resulted in anamnestic BCoV antibody responses at weaning. Clinical relevance: Prime-boost vaccination should be considered for control of BCoV respiratory disease.

Comparaison des réponses en anticorps ELISA neutralisant le virus et spécifiques du virus chez des nouveau-nés bovins vaccinés par amorces-rappels différenciés contre le coronavirus bovin. Objectif: Cette étude a abordé le manque actuel de connaissances sur les réponses immunitaires néonatales de stimulation pour maitriser la maladie respiratoire à coronavirus bovin (BCoV) chez les bovins de boucherie en âge de sevrage. Animaux: Les études 1 et 2 portaient respectivement sur 33 et 22 veaux de boucherie néonatals croisés commerciaux. Procédures: L'étude 1 a comparé les concentrations d'anticorps neutralisant le BCoV de veaux témoins avec 3 groupes de veaux vaccinés de manière différentielle avec des vaccins à virus vivant modifié (MLV) contre le BCoV pour administration par voie mucosale et/ou systémique. L'étude 2 a comparé les concentrations d'anticorps spécifiques et neutralisants parmi des groupes de veaux sensibilisés au BCoV par voie mucosale et qui ont eu un rappel par voie systémique différentielle. Résultats: Dans l'étude 1, les veaux qui avaient reçu une amorce au niveau des muqueuses et un rappel systémique présentaient des concentrations d'anticorps neutralisant le BCoV plus élevées que le groupe témoin au sevrage. Dans l'étude 2, le rappel des veaux amorcés par voie mucosale par l'injection d'un vaccin inactivé ou MLV a entraîné une réponse anamnestique en anticorps spécifiques du BCoV au sevrage. Conclusion: En période néonatale, l'amorce par voie mucosale et le renforcement systémique ont entraîné des réponses anamnestiques en anticorps BCoV au sevrage. Pertinence clinique: La vaccination de rappel doit être envisagée pour maitriser la maladie respiratoire causée par le BCoV.(Traduit par Dr Serge Messier).

Serological investigation and isolation of Salmonella abortus equi in horses in Xinjiang.

BACKGROUND: Salmonella enterica subspecies enterica serovar abortus equi (S. abortus equi) is one of the main pathogens that causes abortion in pregnant horses and donkeys, which was highly infectious and greatly restricts the healthy development of the horse industry. OBJECTIVES: In order to investigate the prevalence and biological characteristics of S. abortus equi in different regions and breeds of horses in Xinjiang. METHODS: This study conducted ELISA detection of S. abortus equi antibodies on serum samples of 971 horses collected from three large-scale horse farms and five free-range horse farms in Yili Prefecture and Bayingol Mongolian Autonomous Prefecture of Xinjiang from 2020 to 2023. On this basis, bacterial isolation, culture, identification, and drug sensitivity tests were conducted on 42 samples of aborted foal tissues and 23 mare vaginal swabs. RESULTS: The results showed that the positive rate of S. abortus equi antibody was as high as 20.91% in 971 horse serum samples. Among them, the positive rate in the Ili region (29.09%) was significantly higher than that in the Bayingole region (11.24%), and the positive rate in mares (22.45%) was higher than that in stallions (14.05%). In terms of horse breeds, the positive rates of self-propagating thoroughbred horses, half-bred horses, Ili horses and Yanqi horses were 43.22%, 28.81%, 14.72% and 11.24% respectively. In addition, S. abortus equi was more susceptible to juvenile and elderly horses, with positive rates of 70.00%and 41.86%, respectively, both of which were significantly higher than young (10.97%) and adult (19.79%) horses. Further, 9 strains of S. abortus equi were obtained through bacterial isolation, culture and identification, which were resistant to five antibiotics (Clarithromycin, Clindamycin, penicillin, Sulfamethoxazole and Rifampicin), and sensitive to 13 antimicrobial agents (Amoxicillin, Ciprofloxacin and Gentamicin, et al.). CONCLUSION: There was a high infection rate of S. abortus equi in Ili Prefecture and self-propagating thoroughbred horses, and juvenile or old mares were more susceptible, which will provide scientific basis for the prevention of S. abortus equi infection in different regions and breeds of horses in Xinjiang.

Post-Vaccination Sero-Monitoring of Peste des Petits Ruminants in Sheep and Goats in Karnataka: Progress towards PPR Eradication in India.

Peste des petits ruminants (PPR) presents economic challenges in enzootic countries impacting small ruminant productivity. The state of Karnataka, India, implemented a mass vaccination campaign in alignment with the PPR-Global Eradication Programme (GEP) and the National Strategic Plan for PPR eradication. This study was conducted from January to March 2023 to assess seroconversion in post-vaccinated goats and sheep at the epidemiological unit (epi-unit) level, aligning with the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) guidelines in the PPR Global Control and Eradication Strategy (GCES). Before vaccination, 3466 random serum samples were collected from small ruminants of three age groups (6-12 months, 1-2 years, and >2 years) across 116 epi-units, spanning 82 taluks in 28 districts. Post-vaccination sero-monitoring included 1102 serum samples collected from small ruminants of the 6-12-month age group only, across 111 epi-units covering 64 taluks in 23 districts. The PPRV antibody status was determined using an indigenous hemagglutinin (H) protein monoclonal antibody-based competitive ELISA kit. Pre-vaccination, the PPR seropositivity rates were 55%, 62%, and 66% in the age groups of 6-12 months, 1-2 years, and >2 years, respectively, with a 61% PPRV antibody prevalence across all the age groups. Notably, 41% of the epi-units exhibited antibody prevalence rates of ≥70%, indicating a substantial population immunity, possibly attributed to the previous vaccination program in the state since 2011. In contrast, only 17% of the epi-units had below 30% seroprevalence rates, emphasizing the need for intensified vaccination. Statistical analysis of the data revealed significant correlations (p < 0.05) between the presence of PPRV antibodies and host factors such as species, breed, and sex. Post-vaccination seroprevalence in the 6-12 months age group was found to be 73.4%, indicating the use of an efficacious vaccine. On the evaluation of vaccination immunity in the 6-12 months age group, it was revealed that over 69% of the epi-units achieved a response surpassing ≥70%, indicating a significant improvement from 42% of the epi-units in pre-vaccination. For active PPR eradication, a mass vaccination campaign (>95% coverage) targeting small ruminant populations aged >4 months is advocated, aiming to achieve the desired herd immunity of >80%. This study offers crucial insights into PPR baseline seroprevalence/immunity status and vaccine efficacy, guiding national strategies towards a PPR-free India and further supporting the global eradication initiative.

Seroprevalence of Infection by Borrelia Species Responsible for Lyme Disease in the French Alps: Analysis of 27,360 Serology Tests, 2015-2020.

Objectives: Lyme borreliosis incidence is increasing in several areas; moreover, it has recently gained the public's attention. Apart from erythema migrans, Lyme disease diagnosis relies (among others) on serology test; however, the prevalence of positive enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay has been poorly studied in the general population. We aimed to approach the seroprevalence of infection by Borrelia species responsible for Lyme disease in the French Isere department using city laboratories data. Patients and Methods: We retrieved all serological tests for Borrelia species responsible for Lyme disease performed in the two main networks of city laboratories between 2015 and 2020. All patients with both ELISA and WB IgG were considered seropositive. Results: We analyzed 27,360 tests (ELISA/ELISA+WB). Mean age was 50.9 ± 20.3 years (ranges: 0-101), with 57.1% females. Overall, 11.7% had IgG detected by ELISA, and 4.7% had IgG detected by both ELISA and WB assay. Seropositive status was more frequent in males (7.0% vs. 2.9%, p < 0.001). Seropositivity rate increased with age after a first peak in childhood; men aged 61-70 years had the highest seropositivity rate (10.3%). In addition, seropositivity rate was higher in persons from a rural area. In multivariate analysis, older age, male gender and living in a rural area were independently associated with seropositivity. Seropositivity rate was stable on the 2017-2020 period. Conclusion: The seroprevalence of infection by Borrelia species responsible for Lyme disease is high in Isere; this probably reduces the predictive positive value for Lyme disease of ELISA and WB IgG, suggesting that this serological test should not be performed for nonspecific symptoms.

Non-specific effects of inactivated Mycobacterium bovis oral and parenteral treatment in a rabbit scabies model.

Tuberculosis BCG vaccination induced non-specific protective effects in humans led to postulate the concept of trained immunity (TRAIM) as an innate type of immune mechanism that triggered by a pathogen, protects against others. Killed vaccines have been considered not to be effective. However, field efficacy of a commercial vaccine against paratuberculosis, as well as of a recently developed M. bovis heat-inactivated vaccine (HIMB) prompted to test whether it could also induce TRAIM. To this, we used a sarcoptic mange rabbit model. Twenty-four weaned rabbits were treated orally or subcutaneously with a suspension of either HIMB (107 UFC) or placebo. Eighty-four days later the animals were challenged with approximately 5000 S. scabiei mites on the left hind limb. Skin lesion extension was measured every 2 weeks until 92 days post-infection (dpi). Two animals were killed at 77 dpi because of extensive skin damage. The rest were euthanized and necropsied and the lesion area and the mite burden per squared cm were estimated. Specific humoral immune responses to S. scabiei and to M. bovis were investigated with the corresponding specific ELISA tests. Subcutaneously and orally HIMB vaccinated animals compared with placebo showed reduced lesion scores (up to 74% and 62%, respectively) and mite counts (-170% and 39%, respectively). This, together with a significant positive correlation (r = 0.6276, p = 0.0031) between tuberculosis-specific antibodies and mite count at 92 dpi supported the hypothesis of non-specific effects of killed mycobacterial vaccination. Further research is needed to better understand this mechanism to maximize cross protection.

Diagnostic performance of Mycoplasmopsis bovis antibody ELISA tests on bulk tank milk from dairy herds.

BACKGROUND: Testing of bulk tank milk (BTM) for Mycoplasmopsis bovis (previously Mycoplasma bovis) antibodies is increasingly popular. However the performance of some commercially available tests is unknown, and cutoff values possibly need to be adjusted in light of the purpose. Therefore, the aim of this study was to compare the diagnostic performance of three commercially available M. bovis antibody ELISAs on BTM, and to explore optimal cutoff values for screening purposes. A prospective diagnostic test accuracy study was performed on 156 BTM samples from Belgian and Swiss dairy farms using Bayesian Latent Class Analysis. Samples were initially classified using manufacturer cutoff values, followed by generated values. RESULTS: Following the manufacturer's guidelines, sensitivity of 91.4%, 25.6%, 69.2%, and specificity of 67.2%, 96.8%, 85.8% were observed for ID-screen, Bio K432, and Bio K302, respectively. Optimization of cutoffs resulted in a sensitivity of 89.0%, 82.0%, and 85.5%, and a specificity of 83.4%, 75.1%, 77.2%, respectively. CONCLUSIONS: The ID-screen showed the highest diagnostic performance after optimization of cutoff values, and could be useful for screening. Both Bio-X tests may be of value for diagnostic or confirmation purposes due to their high specificity.

Bayesian latent class modelling of true prevalence in animal subgroups with application to bovine paratuberculosis infection.

The prevalence of an infectious disease of animals living in separate groups (e.g. herds) is naturally analyzed using a Bayesian hierarchical latent class model. We propose an extension to this methodology by including subgroup level prevalence measures within the groups of animals. As an application illustrating the merits of our methodology, we reassessed the prevalence of bovine paratuberculosis (PTBC) infection in Hungarian commercial dairy farms. Our aim was to consolidate previous findings using a large amount of recent data and priors based on historical data. To model the subgroup level infection prevalence within animal groups, we considered correlated prevalences following beta distributions derived from independent normally distributed random herd effects. In the application, infection status of herds was handled as latent classes, multiparous and primiparous cows as within-herd subgroups. The novel methodology allows us to estimate both the mean and median conditional within-herd true prevalence (CWHP) related to each animal subgroup as well as other measures characterizing the interrelation of subgroups. The results of the application aligned with the findings of the former PTBC study, while the more recent and considerably larger dataset and the use of historical priors increased the reliability of the results. The STAN and JAGS codes of the application are available in Supplementary material.

Development of an indirect ELISA for the serodiagnosis of canine infection by Onchocerca lupi.

Onchocerca lupi is a zoonotic filarioid parasite of dogs and cats with widespread distribution. A specific non-invasive diagnostic assay for the detection of O. lupi infections remains unavailable. This study aimed to assess the accuracy, specificity, and sensitivity of an ELISA test designed using nine peptides from two O. lupi proteins. Sera (n = 54) collected from O. lupi infected dogs from endemic areas (Portugal and USA), alongside sera from dogs positive for Dirofilaria immitis, D. repens, Cercopithifilaria bainae, and Acanthocheilonema reconditum (n = 53) from a non-endemic area for O. lupi, as well as from helminth-free dogs (n = 60), were tested. The checkerboard titration method was applied for the optimization of peptide concentrations and conjugate anti-dog dilutions. Sensitivity, specificity, and optimal cut-off values were calculated using ROC curve analysis. All peptides reacted against sera of O. lupi, with no correlation between optic density (OD) values and microfilariae (mfs) loads. Sensitivity and specificity values ranging from 85.45 to 100%, and 88.89% to 100%, respectively, were recorded for all peptides examined, with 100% specificity and sensitivity observed for peptides 40_3, 40_5, 130_3, 120_3 and 40_1, 130_5, respectively. The maximum cut-off value was observed for peptides 40_5 (0.765) and 40_3 (0.708). Testing of sera from dogs positive for other filarioids resulted in lower OD values (up to 1.565) for peptides 40_3 and 40_5 when compared with O. lupi (up to 2.929). The availability of this assay will be of value in epidemiological studies of canine O. lupi infection in both endemic and non-endemic areas, and in assessing the risk for zoonotic transmission.

A Bayesian finite mixture model approach to evaluate dichotomization method for correlated ELISA tests.

In diagnostic accuracy studies, a commonly employed approach involves dichotomizing continuous data and subsequently analyzing them using a Bayesian latent class model (BLCM), often relying on binomial or multinomial distributions, rather than preserving their continuous nature. However, this procedure can inadvertently lead to less reliable outcomes due to the inherent loss of information when converting the original continuous measurements into binary values. Through comprehensive simulations, we demonstrated the limitations and disadvantages of dichotomizing continuous biomarkers from two correlated tests. Our findings highlighted notable disparities between the true values and the model estimates as a result of dichotomization. We discovered the crucial significance of selecting a reference test with high diagnostic accuracy in test evaluation in order to obtain reliable estimates of test accuracy and prevalences. Our study served as a call to action for veterinary researchers to exercise caution when utilizing dichotomization.

[Prevalence of paratuberculosis in Thuringian sheep and goat flocks]. / Verbreitung der Paratuberkulose in Thüringer Schaf- und Ziegenherden.

OBJECTIVE: In Germany, only few data on the current distribution of paratuberculosis in sheep and goat flocks is available. The present study provides an overview of the distribution of Mycobacterium avium ssp. paratuberculosis (MAP) in 165 Thuringian sheep and goat flocks. Also, the study investigated the association between the MAP status of the flock and herd specific factors as well as the association between the individual measured value of ELISA and animal specific factors like age, body condition, sex, and animal species. MATERIAL AND METHODS: To investigate the prevalence of MAP, serum samples from 2550 sheep and 1171 goats from 165 flocks (flock size 2 to 2879 animals) were serologically examined for MAP antibodies in 2021. Additionally, 1 to 6 environmental faecal samples were collected from every flock depending on the flock size. They were examined for the presence of MAP by using both bacteriological cultivation and a commercially available real-time-PCR. RESULTS: MAP antibodies were detected in 41 sheep (1.6%) and 29 goats (2.5%), which accounts to a detection of MAP antibodies in 20.6% of the 165 flocks (on herd level). The symptoms of paratuberculosis, weight loss with preserved appetite and altered fecal consistency, were observed in only four of the flocks. A positive association was identified between the detection of MAP or MAP-specific antibodies in a flock and flock size, as well as positive association between the measured value in the Elisa (s/p ratio) and the age of the animal. Furthermore, an association between an increasing s/p ratio of the ELISA and a decreasing body condition was found. CONCLUSION AND CLINICAL RELEVANCE: Given what is known about the distribution of paratuberculosis in small ruminants, this disease should always be considered as a possible cause of weight loss and diarrhea. In case of high within-herd prevalence herd-specific control measures should be considered. In serological herd monitoring, animals with poor body condition should preferably be included in the sample, as the probability of being able to identify MAP positive animals is higher here.

Design and development of multiepitope chimeric antigens by bioinformatic and bacterial based recombinant expression methods, with potential application for bovine tuberculosis serodiagnosis.

Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis, is a single health concern, which causes economic losses, is a sanitary barrier and is a zoonotic concern. The golden-pattern intradermic tests have low sensitivity of about 50%. To fix this sensitivity problem, immunoassays could be a powerful tool. However, few studies produced antigens for bTB immunoassays, which needs improvements. Aim of this study was to produce multiepitope chimeric antigens (MCA) to use for bTB diagnosis. To achieve MCA design and development, extensive bibliographic search, antigenic epitope prediction, specificity, hydrophobicity, and 3D structure modeling analyses were performed, as well as cloning, expression and purification. Seven epitopes from four different target proteins (MPB-70, MPB-83, ESAT-6 and GroEL) were combined in five chimeras containing five repetitions of each epitope to enhance antibodies affinity. 3D predicted models revealed that all chimeras have a high percentage of disorder, which could enhance antibody recognition, although taking to protein instability. Each chimera was cloned into pET28a (+) expression plasmids and expressed in six Escherichia coli expression strains. Chimeras 3, 4 and 5 could be solubilized in 8 M urea and purified by ion exchange affinity chromatography. Against bTB positive and negative sera, purified chimera 5 had the best results in indirect dot blot and ELISA, as well as in lateral flow dot blot immunoassay. In conclusion, chimera 5, an MPB-83 containing MCA, could be used for further studies, aimed to develop a serologic or rapid test for bTB diagnosis.

Serum symmetric dimethylarginine in older dogs: Reference interval and comparison of a gold standard method with the ELISA.

BACKGROUND: Serum symmetric dimethylarginine (SDMA) is used to screen for renal dysfunction in dogs. The gold standard technique for measuring SDMA, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is not widely available. Age-specific reference intervals for SDMA in older dogs are lacking. OBJECTIVES: Prospective study in older dogs to validate a commercially available LC-MS/MS method for SDMA, compare SDMA concentrations with concentrations measured using ELISA and obtain a reference interval (RI) for older dogs using both methods. ANIMALS: Client-owned older dogs undergoing health screening. METHODS: The LC-MS/MS method was analytically validated (limit of detection, precision, and linearity). Serum was sent cooled overnight for ELISA or was frozen at -80°C until batch analysis using LC-MS/MS. Results of LC-MS/MS and ELISA were compared and RIs for older dogs were calculated according to international guidelines. RESULTS: The LC-MS/MS method showed good linearity (r2 = .99) and precision (coefficient of variation <10%), with a laboratory RI between 8.0 and 14.0 µg/dL. Paired measurements were available from 118 different dogs. Median SDMA concentration were 9.4 (range, 5.0-21.2) using LC-MS/MS and 12.0 (range, 5.0-22.0) µg/dL using ELISA. Both methods significantly differed with a mean difference of 2.2 µg/dL. The RI for older dogs for LC-MS/MS was 4.4-15.0 µg/dL, and for ELISA was 6.4-17.4 µg/dL. CONCLUSIONS AND CLINICAL IMPORTANCE: The ELISA provided significantly higher SDMA concentrations compared to the validated LC-MS/MS method, indicating the need for device- or assay-specific RI. The obtained age-specific RI for SDMA is considerably higher in older dogs compared to the general laboratory RI.

Evaluation of LAMP for Fasciola hepatica detection from faecal samples of experimentally and naturally infected cattle.

Fasciola hepatica causes liver fluke disease in production animals and humans worldwide. Faecal egg counts (FEC) are the most common diagnostic tool for the diagnosis of liver fluke disease. However, FEC has low sensitivity and is often unreliable for the detection of patent infection. In this study, loop-mediated isothermal amplification (LAMP) was optimised and evaluated for the detection of Fasciola hepatica infection, with the aim of increased sensitivity and making it suitable for on-farm application. LAMP was initially conducted under laboratory conditions, optimised to enable visual detection using calcein dye. DNA extraction based on bead-beating was developed to enable on-farm application. LAMP results were compared to FEC and polymerase chain reaction (PCR). Under laboratory conditions, LAMP was conducted using two incubation methods: a conventional PCR thermocycler and a field-deployable LAMP instrument. When compared to a 'rigorous' FEC protocol consisting of multiple counts using a comparatively large volume of faeces and with infection confirmed post-mortem, LAMP was highly sensitive and specific (using silica membrane DNA extraction sensitivity 88 %, specificity 100 %; using sieving and beat-beating DNA extraction sensitivity 98.9 %, specificity 100 %). When applied on-farm, LAMP was compared to conventional FEC, which suggested high sensitivity but low specificity (sensitivity 97 %, specificity 37.5 %). However, further analysis, comparing field LAMP results to laboratory PCR, suggested that the low specificity was likely the outcome of the inability of conventional FEC to detect all true F. hepatica positive samples. Based on the high sensitivity and specificity of LAMP compared to a 'rigorous' FEC protocol and its ability to be used in field settings, the study demonstrates the potential of LAMP for diagnosing F. hepatica infection in agriculture.

Determination of expression patterns of miR-26a, and preimplantation factor levels for early pregnancy detection in nulliparous and multiparous cows.

For maximum productivity in a dairy farm, the earliest and the most accurate detection of pregnancy is essential. The aim of this study was to determine the efficacy of expression patterns of miR-26a, and serum Preimplantation Factor (PIF) levels for pregnancy diagnosis during the early pregnancy in nulliparous and multiparous cows. A total of 60 cows (30 nulliparous and 30 multiparous Holstein cows) were enrolled in the study. Blood samples were collected for miR-26a on days 8 and 16 (D8 and D16), and for the PIF on days 10 and 20 (D10 and D20) following insemination (D0). Pregnancies were determined by ultrasonography on the 28th day after insemination. Expression levels of miR-26a determined by qPCR. PIF levels were assessed by using commercial ELISA kits. All data were analyzed by using the MIXED procedure of SPSS. The expression levels of miR-26a were 6.64 folds higher on D16 in pregnant compared to non-pregnant multiparous cows (p < .05). On D8 and D16, miR-26a expression levels were found higher 13 folds in pregnant compared to non-pregnant nulliparous cows (p < .05). Additionally, miR-26a expressions were higher 5.42 folds (p < .05) on D8, 7.19 folds higher (p < .01) on D16 in pregnant nulliparous and multiparous cows, and were 6.30 folds higher (p < .001) on D8 and D16 according to non-pregnant animals. PIF levels were greater in pregnant animals (p < .05). Analyzing miR-26a on D8 might be considered as sufficient in nulliparous cows. Pregnancy detection in multiparous cows can be made on the 16th day with this method. Furthermore, PIF evaluations may be sufficient on D10 in multiparous cows. Besides, PIF levels and miR-26a expression levels might be used safely in field conditions and clinical applications.

Comparison of bovine viral diarrhea virus detection methods: Results of an international proficiency trial.

Bovine viral diarrhea virus (BVDV), one of the most important infectious cattle diseases globally, is being combated in multiple countries. The main source for virus transmission within herds and especially to unaffected cattle farms are life-long persistently infected (PI), immunotolerant animals. Therefore, the early identification of PI calves is a major pillar of disease control programs. In addition, rapid and reliable virus identification is necessary to confirm the causative agent in acute clinical cases. Here, we initiated an international interlaboratory proficiency trial in order to evaluate BVDV detection methods. Four ear notch samples and four sera were provided to the participating veterinary diagnostic laboratories (n = 40). Two of the ear notches and two sera contained BVDV and two ear notches and one serum were negative for pestiviruses. The remaining serum was positive for the ovine border disease virus (BDV). The sample panel was analyzed by an ERNS-based ELISA for antigen detection, diverse real-time RT-PCR (RT-qPCR) assays and/or virus isolation. Occasionally, additional typing of the virus strains was performed by sequencing or specific antibody staining of the obtained cell culture isolates. While the antigen ELISA allowed reliable BVDV diagnostics, infectious virus could be isolated only in just under half of the attempts (43.33%). RT-qPCR enabled the sensitive detection of pestiviruses, though an impact of the extraction method on the resulting quantification cycle values was observed. In general, subsequent typing of the detected virus strains is required to differentiate BVDV from BDV infections. In conclusion, for BVDV identification in clinical cases or in the context of disease control, RT-qPCR methods or ERNS antigen ELISAs should be preferentially used.

A novel Bayesian Latent Class Model (BLCM) evaluates multiple continuous and binary tests: A case study for Brucella abortus in dairy cattle.

Bovine brucellosis, primarily caused by Brucella abortus, severely affects both animal health and human well-being. Accurate diagnosis is crucial for designing informed control and prevention measures. Lacking a gold standard test makes it challenging to determine optimal cut-off values and evaluate the diagnostic performance of tests. In this study, we developed a novel Bayesian Latent Class Model that integrates both binary and continuous testing outcomes, incorporating additional fixed (parity) and random (farm) effects, to calibrate optimal cut-off values by maximizing Youden Index. We tested 651 serum samples collected from six dairy farms in two regions of Henan Province, China with four serological tests: Rose Bengal Test, Serum Agglutination Test, Fluorescence Polarization Assay, and Competitive Enzyme-Linked Immunosorbent Assay. Our analysis revealed that the optimal cut-off values for FPA and C-ELISA were 94.2 mP and 0.403 PI, respectively. Sensitivity estimates for the four tests ranged from 69.7% to 89.9%, while specificity estimates varied between 97.1% and 99.6%. The true prevalences in the two study regions in Henan province were 4.7% and 30.3%. Parity-specific odds ratios for positive serological status ranged from 1.2 to 2.2 for different parity groups compared to primiparous cows. This approach provides a robust framework for validating diagnostic tests for both continuous and discrete tests in the absence of a gold standard test. Our findings can enhance our ability to design targeted disease detection strategies and implement effective control measures for brucellosis in Chinese dairy farms.

Development of an indirect ELISA system for diagnosis of porcine edema disease using recombinant modified Stx2e antigen.

AIM: This study aimed to develop a sensitive and specific recombinant antigen protein indirect enzyme-linked immunosorbent assay (ELISA) kit to detect the Shiga toxin (Stx)-producing Escherichia coli (STEC) antibodies against porcine edema disease (ED). METHODS AND RESULTS: The recombinant antigen was co-expressed with the STEC-derived Stx2e A2-fragment and Stx2e B protein in E. coli BL21(DE3) pLysS cells and purified using maltose-binding protein open columns. We used a Shiga-like toxin 2 antibody to test the specificity of the recombinant antigen in an indirect ELISA, which was detected in antigen-coated wells but not in uncoated wells. We tested the indirect ELISA system using samples from the STEC-immunized pig group, the commercial swine farm group, and healthy aborted fetal pleural effusion group; five and twenty samples, respectively, were positive for STEC in the former, whereas all three samples were negative for STEC in the latter. CONCLUSIONS: This newly developed indirect ELISA may be a specific method for diagnosing STEC infections in pigs.

Serological monitoring of Enterococcus cecorum specific antibodies in chickens.

Pathogenic Enterococcus cecorum (EC) has gained increasing importance as the cause of skeletal infections in meat-type chicken production. Since effective intervention strategies are scarce, it must be focused on preventive measures. Vaccination of meat-type breeder chicken flocks is common practice to protect the progeny against infection with EC. However, no data are available on seroconversion after infection or vaccination. The aim of the present study was the serological monitoring of chickens for EC-specific immunoglobulin Y (IgY) using a newly established EC-specific, indirect ELISA for chickens. Sera from previous infection studies were used for the establishment of the assay. Serum samples from confirmed EC-positive meat-type chicken flocks, vaccinated, and non-vaccinated meat-type chicken breeder flocks were analyzed for EC-specific IgY. Comparison of ELISA results with results from real-time PCR and/or bacteriological examination via culture revealed fair to substantial agreement. In infected chickens, more samples were classified as positive via ELISA than via real-time PCR and/or bacteriological examination via culture. Focusing on chickens experimentally infected at 1 day post-hatch (dph), the highest proportion of positive results and highest S/P ratios were found at 42 dph (p < 0.05). A similar trend was observed for the samples from naturally infected chickens (p < 0.05). Adjustment of the secondary antibody against immunoglobulin M (IgM) may open possibilities to use the assay during the early phase of the growing period, when there is still a chance to treat the infection. The examination of samples from vaccinated and non-vaccinated meat-type breeder chickens revealed no significant differences of S/P ratios independent of farm and autogenous vaccine used. In addition to that, monitoring of a non-vaccinated meat-type breeder chicken flock at 4, 10, 15, and 19 weeks post-hatch showed a continuous increase of ELISA-positive serum samples associated with an increase of S/P ratios. This may be explained by cross reactivity with antibodies to Enterococcus hirae or natural antibodies. The usage of EC-specific, recombinant proteins for coating of the plates may help to reduce unspecific background and increase the assay's specificity in future applications. In conclusion, the newly developed ELISA provides a suitable tool for serological monitoring of meat-type chickens during experimental studies with EC under standardized conditions. Remarkably, the assay is able to detect a higher proportion of EC-positive chickens than other methods, which are currently available. However, the assay is not yet suitable for the monitoring of breeder flocks due to high background.