Determination of colostrum quality using Brix refractometer in sheep.

In this study, we aimed to determine the quality of colostrum in sheep by using Brix refractometer. The research included 100 sheep of Merino X Kivircik crossbred. From each, we collected 15 mL of colostrum samples in falcon tubes within the first 8 h after delivery. Mean colostral IgG level of sheep was 156.68 ± 7.23 g L-1, optical and digital Brix refractometer values (%) were determined as 27.43 ± 0.53 and 27.69 ± 0.60, respectively. Ewes carrying twin lambs produced significantly higher quality colostrum than those carrying single lambs. However, parity did not affect the colostrum quality. Optical and digital Brix values were correlated with gold standard radial immunodiffusion (RID) colostral IgG level (r = 0.70 and r = 0.64, respectively). Also, optical and digital Brix refractometers were found to be highly correlated (r = 0.98, P < 0.001). While the optimal Brix value was 22% for the 50, 60 and 70 g L-1 IgG threshold values (by means of RID as the potential good quality threshold value for ewe colostrum quality), this value was 23% for 80 g L-1. We can conclude that Brix refractometers is a valuable tool for determining ewe colostrum quality. A cut point of 22% Brix for defining good quality colostrum in ewes was most appropriate for our data.

Deciphering immunodiffusion: In silico optimization for faster protein diagnostics.

Immunodiffusion tests offer a simple yet powerful method for detecting protein antigens, but their long assay times hinder clinical utility. We unveil the complex interplay of parameters governing this process using finite element simulations. By meticulously validating our model against real-world data, we elucidate how initial concentrations and diffusivities of antigen and antibody shape the intensity, size, and formation time of the precipitin ring. Our key innovation lies in employing phase diagram analysis to map the combined effects of these parameters on assay performance. This framework enables rapid in silico parameter estimation, paving the way for the design of novel immunodiffusion assays with drastically reduced assay times. The presented approach holds immense potential for optimizing protein diagnostics for fast and reliable diagnostics.

Evaluation of the Analytical Performance of a Lateral Flow Assay for the Detection of Anti-Coccidioides Antibodies in Human Sera-Argentina.

Coccidiomycosis is a potentially life-threatening fungal infection endemic to certain regions of Argentina. The infection is caused by Coccidioides spp. and is primarily diagnosed by Coccidioides antibody (Ab) detection. Access to rapid, highly accurate diagnostic testing is critical to ensure prompt antifungal therapy. The sona Coccidioides Ab Lateral Flow Assay (LFA) performs faster and requires less laboratory infrastructure and equipment compared with other Ab detection assays, potentially providing a substantial improvement for rapid case screening in coccidioidomycosis-endemic regions; however, validation of this test is needed. Thus, we aimed to evaluate the analytical performance of the sona Coccidioides Ab (LFA) and compare agreement with anti-Coccidioides Ab detection assays. A total of 103 human sera specimens were tested, including 25 specimens from patients with coccidioidomycosis and 78 from patients without coccidioidomycosis. The sona Coccidioides Ab Lateral Flow Assay (LFA) was performed with a sensitivity of 88%, and specificity and accuracy of 87%. Furthermore, the Coccidioides Ab LFA had good agreement with other anti-Coccidioides Ab detection assays. Our findings suggest the sona Coccidioides Ab LFA has satisfactory performance and may be useful for diagnosing coccidioidomycosis in endemic regions.

Immunity transfer in mule foals fed with good IgG quality colostrum.

While the passive transfer of immunity in horse and donkey foals has been extensively studied, there is limited information for mule foals. Immunoglobulin type G (IgG) and serum total protein concentration (TP) were assessed at different sampling times to evaluate the correlation between serum radial immunodiffusion (SRID) with electrophoresis, refractometry, and dry chemistry analyzer (Biuret), and to estimate serum IgG concentrations using serum TP in mule foals. We analyzed a total of 30 samples collected at birth, and at 6, 12, 24, and 48 h of life from 6 mule foals by SRID, electrophoresis TP, biuret TP, and refractometry TP. The SRID IgG concentration significantly increased from birth until T6 (p < 0.001). Serum TP analyzed with refractometry revealed differences between T0 and T12, T24 and T48 (p < 0.05), while a significant difference was observed with the biuret method between T0 and all the other sampling times (p < 0.001). A strong correlation was found between IgG SRID and biuret TP (r = 0.69, p < 0.001), and a good correlation existed between IgG SRID, refractometry TP, and electrophoresis TP (r = 0.44, p < 0.01 and r = 0.39, p < 0.05, respectively). All methods can be used to estimate the passive transfer of immunity in mule foals. TP refractometry and biuret TP values can be used to determine serum IgG concentrations in the blood of mule foals on their first day of life through the application of a specific equation.

Establishment of Reference Reagents for Single-Radial-Immunodiffusion Assay on the 2022/23 Seasonal Influenza Vaccine in Japan and Their Quality Validation.

Potency tests for influenza vaccines are currently performed using a single-radial immunodiffusion (SRID) assay, which requires a reference antigen and anti-hemagglutinin (HA) serum as reference reagents. Reagents must be newly prepared each time a strain used for vaccine production is modified. Therefore, establishing reference reagents of consistent quality is crucial for conducting vaccine potency tests accurately and precisely. Here, we established reference reagents for the SRID assay to conduct lot release tests of quadrivalent influenza vaccines in Japan during the 2022/23 influenza season. The potency of reference antigens during storage was confirmed. Furthermore, we evaluated the cross-reactivity of each antiserum raised against the HA protein of the 2 lineages of influenza B virus toward different lineages of influenza B virus antigens to select a suitable procedure for the SRID assay for accurate measurement. Finally, the intralaboratory reproducibility of the SRID assay using the established reference reagents was validated, and the SRID reagents had sufficient consistent quality, comparable to that of the reagents used for testing vaccines during previous influenza seasons. Our study contributes to the quality control of influenza vaccines.

Comparison of coccidioidal complement fixation and quantitative immunodiffusion serology at a reference laboratory.

The incidence of coccidioidomycosis continues to increase. The diagnosis frequently relies on non-invasive diagnostic testing with immunodiffusion and complement fixation (CF) testing the current gold standard. A direct comparison of quantitative immunodiffusion and CF for IgG antibodies has not been previously reported. In a comparison of 368 samples, there was close concordance observed (360/368 = 97.8%) (P-value < .001). These tests can be considerably interchangeable in the reference laboratory setting.

There are several diagnostic methodologies available in coccidioidomycosis. Direct comparisons of these methods are limited. Prior studies have not compared quantitative immunodiffusion to complement fixation testing. Our results show these tests are highly concordant.

Evaluation of commercial immunodiffusion reagents for detecting serum anti-Paracoccidioides antibodies

ABSTRACT Background: Accurate diagnosis of paracoccidioidomycosis is crucial for improving patient outcomes. Paracoccidioides antibody detection by double immunodiffusion (DID) is a convenient diagnostic tool, but testing performance can vary based on certain factors. Methods: We assessed DID performance using a commercially prepared Paracoccidioides reagents (IMMY, USA), involving 40 serum specimens, including 20 from patients with proven paracoccidioidomycosis and 20 from patients without the disease. The DID test demonstrated a sensitivity of 90% (95% CI=68%-99%) and a specificity of 100% (95% CI=83%-100%). Conclusions: Our findings suggest that DID using commercial reagents may provide a feasible tool with satisfactory testing performance for anti-Paracoccidioides antibody detection.

Investigation of brix refractometry for estimating bovine colostrum immunoglobulin G concentration.

Many dairy operations uses a Brix refractometer to assess the quality of first-milking colostrum. This study investigated whether a digital Brix refractometer could be used in a model to predict colostrum IgG concentration and whether more than one %Brix threshold could be used for different colostrum IgG concentrations. Colostrum from 182 animals was tested using a digital Brix refractometer and by single radial immunodiffusion. Statistical analysis, using simple linear regression to relate %Brix results with corresponding colostral IgG concentration, and receiver operating characteristic (ROC) analysis were used to identify %Brix cutoffs that had no false positive results. Colostral IgG concentrations from digital Brix refractometry had a R2 value of 0.818 and a S-value of 21.7 g/L. The large S-value shows that a digital Brix refractometer should not be used in a model to predict colostrum IgG concentration. However, %Brix scores of 19.0, 22.0, 25.0 and 30.0 percent can be used to estimate minimum colostral IgG concentrations of 25, 50, 75 and 100 g/L. These four cutoffs can be used to strategically feed smaller volumes of colostrum to newborn calves. Smaller volumes may reduce unwanted side effects and shorten the time interval in which calves refuse to nurse, while still delivering an adequate mass of IgG to have successful transfer of passive immunity.

Immunogenic characterization of AlPO4 adsorbed Td vaccine and liposome-mediated Td vaccine.

Purpose: The purpose of this study was to compare the antigenic potency and stability of tetanus and diphtheria (Td) vaccines when combined with aluminum phosphate (AlPO4) and liposome adjuvants. Materials and Methods: In vitro and in vivo analyses were conducted using the single radial immunodiffusion method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Td vaccines were prepared with AlPO4 adsorption and liposome-mediated delivery, and protein antigens were characterized using these methods. Results: The results revealed that the liposome-mediated Td vaccines exhibited higher immunogenicity compared to the AlPO4-adsorbed Td vaccines. Additionally, the liposome-mediated Td vaccines demonstrated higher stability as native antigens. Conclusion: This study highlights the importance of utilizing liposome adjuvants in vaccine development. The liposome-mediated Td vaccines showed enhanced immunogenicity and stability, making them a promising approach for improving vaccine efficacy. Understanding and optimizing adjuvant strategies can contribute to the development of effective vaccines against various diseases.

Paracoccidioidomycosis cases by Paracoccidioides lutzii in southeastern Brazil.

BACKGROUND AND OBJECTIVE: Paracoccidioidomycosis (PCM) is a systemic fungal disease caused by the thermodimorphic fungi Paracoccidioides spp. Their distribution is highly variable. Paracoccidioides lutzii is predominantly found in North and Middle-West Brazil and Ecuador. This study evaluated the clinicopathological characteristics of 10 patients diagnosed with PCM caused by P. lutzii in a reference center located in southeastern Brazil. DESIGN: Double immunodiffusion assay (DID) was used to investigate 35 patients' sera with negative serology for P. brasiliensis against a P. lutzii CFA (cell-free antigen). RESULTS: Out of the 35 retested patients, 10 (28.6%) were positive for P. lutzii CFA. Four patients did not report any displacement to P. lutzii endemic areas. Our results reinforce the importance of using different antigens when testing patients with clinical manifestations of PCM and negative serological tests for P. brasiliensis, primarily in cases of the report of displacement to or former residence in P. lutzii endemic regions. CONCLUSIONS: The availability of tests for different Paracoccidioides species antigens is fundamental for reaching an adequate diagnosis, patient follow-up, and definition of prognosis.

Comparison of 4 agar gel immunodiffusion kits for serologic detection of equine infectious anemia virus antibodies.

Using 85 sera collected from horses that had been experimentally infected with equine infectious anemia virus (EIAV) and 200 field sera collected from racehorses in Japan, we compared 4 agar gel immunodiffusion (AGID) kits for serologic detection of EIAV antibodies from Idexx, VMRD, IDvet, and the National Engineering Research Center of Veterinary Biologics, China (NECVB). The positive control lines were sufficiently clear in all kits for evaluation to be made, with slight differences in sharpness: NECVB was the sharpest, followed by VMRD, IDvet, and Idexx. The test results for all 285 samples agreed among the 4 kits, with 62 positives and 223 negatives. The sensitivities and specificities of VMRD, IDvet, and NECVB compared with the Idexx kit were 100%, and the kappa coefficient values between the kits were 1.0 for all combinations. We concluded that the testing capacity of these 4 kits was virtually identical.

Review of Clinical and Laboratory Diagnostics for Coccidioidomycosis.

Coccidioidomycosis is a fungal disease associated with soil exposure that frequently goes undiagnosed due at least in part to its nonspecific presentation and the lack of clinical suspicion by health care providers. Currently available diagnostics for coccidioidomycosis offer qualitative results that can suffer from low specificity, while semiquantitative assays are labor-intensive and complex and can require multiple days to complete. Furthermore, significant confusion exists regarding the optimal diagnostic algorithms and appropriate usage of available diagnostic tests. This review aims to inform clinical laboratorians and treating clinicians about the current diagnostic landscape, appropriate diagnostic strategies, and future diagnostic directions for coccidioidomycosis, which is expected to become more prevalent due to increased migration into areas of endemicity and climate changes.

Application progress of high performance liquid chromatography in detection of hemagglutinin content in influenza vaccine / 中国生物制品学杂志

@#At present,the most commonly used method for detecting hemagglutinin(HA)content in influenza vaccines is still single-radial immunodiffusion(SRID). However,the preparation of standards required by this method takes a long time,usually 2 ～ 3 months. Therefore,how to quantitatively analyze HA accurately has always been a difficult problem in the detection of HA content in the situation that reference products can not be obtained at the early stage of the pandemic influenza. High performance liquid chromatography(HPLC)has its own characteristics of rapidity,high sensitivity,good repeatability and high accuracy,which can rapidly determine HA content by using different separation principles and has been widely used in the detection of HA content in influenza vaccine. This paper reviewed the research progress of the application of HPLC in the determination of HA content in influenza vaccine.

Study the Profile of Foot-and-mouth Disease Virus Protein by Electrophoresis and Identification of the Immunodominant Proteins.

Foot-and-mouth disease is an extremely infectious and occasionally fatal viral disease with a rapid onset and a short course that affects cloven-hoofed animals and results in considerable financial losses. Today, Foot-and-mouth disease is controlled by traditional inactivated vaccines. Due to the short duration of immunity, a study was conducted for proteins of the virus as well as obtaining immunodominant proteins to design more efficient vaccines against Foot-and-mouth disease virus. This research aims to study the profile of Foot-and-mouth disease virus protein by electrophoresis and identification of the immunodominant proteins. The purified Foot-and-mouth disease virus was purchased then the protein concentration of that solution was measured by Lowry method. SDS-PAGE was done to achieve the protein profiles of the virus and immunization of 5 guinea pigs was done, then blood samples were taken for obtaining serum. Finally, serology tests; double immunodiffusion, ELISA, and western blotting were used to evaluate antigen response to antibodies (antigenic immunization). The protein concentration was 3.5 mg/ml. In SDS-PAGE with 10% gel, the protein profile of the virus was observed. After immunization, by conducting double immunodiffusion tests, the sediment lines between the serum antibody and the antigen of the virus were formed. Also, The ELISA test showed that the antibodies were formed against the antigens. In the western blot test, two immunodominant proteins of the FMD virus were obtained. According to the results, the immunodominant proteins of the FMD virus were determined. These proteins can be used in immunological diagnostic methods and also novel vaccines.

Anti-Aspergillus fumigatus IgG in patients with bronchiectasis and its relationship with clinical outcome.

Aspergillosis is a mycosis, most commonly affecting the airways. This mycosis can worsen the clinical condition of patients with concurrent lung diseases. We assayed for the presence of serum anti-A. fumigatus IgG in bronchiectasis patients from a tertiary hospital in south Brazil and evaluated the relationship with clinical outcome. Thirty-one patients with bronchiectasis, without cystic fibrosis, were included. Clinical and epidemiological data were collected from all participants. Positive serological tests were detected in 13% (4/31) of the patients. The mortality rate for the year following the assay was, in the seropositive group, 75% (3/4), whereas in the seronegative group, 15% (4/27). An illustrative case is also shown and discussed. Our study highlights the diagnostic challenge and the possible impact of Aspergillus infection on these patients, indicating the necessity of more and larger investigations in the field.

Seroprevalence and risk factors associated with equine infectious anemia in the state of Goiás, Brazil.

Equine infectious anemia (EIA) is an infectious disease affecting equine in most countries and represents a notifiable disease with compulsory euthanasia of positive animals. The present study aimed to determine the prevalence of EIAV infected equines in herds of the state of Goiás (Central Brazil) and to evaluate the risk factors associated with the occurrence of the disease. Blood samples were collected from 1170 equids from 332 randomly selected farms divided into three different strata according to their herd characteristics. Also, an epidemiological questionnaire was applied during the visit to the farm. Of the 332 farms evaluated, 12 (3.1%; 95% CI: 1.24 - 6.00) had at least one positive equine for EIA, and of the 1170 evaluated equines, 14 (2%; 95% CI: 0.31-3.00) were positive in agar gel immunodiffusion. Multivariate analysis revealed that the use of a vaccination pistol (p < 0.001) and the presence of water bodies inside the farm (p < 0.01) were risk factors associated with the occurrence of EIA. Thus, the present study demonstrated a low but widespread prevalence of EIAV infected animals in the herds of Goiás state and that iatrogenic and environmental risk factors were associated with the occurrence of the disease.

Histoplasma capsulatum Complement Fixation and Immunodiffusion Assay Sensitivity in Culture-Confirmed Cases of Histoplasmosis: a 10-Year Retrospective Review (2011 to 2020).

The detection of antibodies against Histoplasma capsulatum remains a frequently relied-on approach to diagnose histoplasmosis. We retrospectively assessed the performances of complement fixation (CF) and immunodiffusion (ID) assays for anti-Histoplasma antibody detection in patients with culture-confirmed histoplasmosis at Mayo Clinic (Rochester, MN) over a 10-year period (2011 to 2020). Among 67 culture-confirmed patients who also had H. capsulatum CF/ID testing ordered, 51 (67.1%) were immunocompromised, 34 (50.7%) had localized disease, and 51 (76.1%) presented with <3 months of symptoms before testing. H. capsulatum CF and/or ID testing was positive in 47 (70.1%) patients, with both assays being positive in 39 cases. CF was positive in 44 (65.7%) patients, with reactivity against both H. capsulatum mycelial and yeast antigens in 30 (68.2%) cases, whereas 11 (25%) and 3 (6.8%) individuals had antibodies to the CF yeast or mycelial antigen only, respectively. H. capsulatum ID was positive in 42 (62.7%) patients, with the presence of the M-band only or the H- and M-bands in 27 (64.3%) and 15 (35.7%) cases, respectively. Among 18 serially tested patients, 12 remained ID and/or CF positive at the final time point (median, 154 days; range, 20 to 480 days). Serial CF testing showed that antibodies to the mycelial antigen serorevert to negative more frequently (6/11) than antibodies to the yeast antigen (2/13). There was no statistically significant difference in antibody positivity relative to patient immune status, degree of disease dissemination, or symptom duration. Serologic testing remains a valuable asset to support the diagnosis of histoplasmosis, particularly when direct detection methods fail to identify an infection.

Comparison of Immunodiagnostic Assays for the Rapid Diagnosis of Coccidioidomycosis in Dogs.

Coccidioidomycosis is a disease caused by the dimorphic fungi Coccidioides spp., which affects humans and a variety of animal species, including domestic dogs. In dogs, accurate diagnosis could provide a substantial improvement on the quality of canine life, as well as an advancement in the mapping of regions endemic for coccidioidomycosis. The purpose of this study was to compare immunodiagnostic assays for anti-Coccidioides antibody (Ab) detection in dogs' serum. Three commercially available immunodiagnostic assays (IMMY®; Norman, OK, USA) were evaluated, including the sona Coccidioides Ab Lateral Flow Assay (LFA), Coccidioides IDCF immunodiffusion assay (IDCF), and the Clarus Coccidioides Ab Enzyme Immunoassay (EIA). Assays were evaluated using 98 dog serum samples: 29 from dogs with coccidioidomycosis, 15 from dogs diagnosed with histoplasmosis, 10 from dogs diagnosed with blastomycosis, and 44 from dogs without a fungal disease. Using specimens from dogs with coccidioidomycosis, the IDCF had an accuracy of 92% (95% confidence interval [95% CI] = 85-96%), the EIA had an accuracy of 91% (95% CI = 83-96%), and the LFA displayed an accuracy of 82% (95% CI = 73-89%). Using Kappa analysis, the agreement between LFA and EIA was 0.59 (95% CI = 0.42-0.75), that between LFA and IDCF was 0.64 (95% CI = 0.48-0.79), and that between EIA and IDCF was 0.79 (95% CI = 0.64-0.90). Most cross-reactions were observed in dogs with histoplasmosis. Compared with EIA and IDCF, the LFA requires substantially less laboratory equipment and infrastructure and rapidly produces results, offering a substantial improvement for the initial screening of coccidioidomycosis in dogs.

False positive anti-Topoisomerase I (Scl-70) antibody results in clinical practice: A case series from a scleroderma referral center.

PURPOSE: To determine if some patients who tested positive for anti-Scl-70 antibody in clinical practice, but did not have classifiable systemic sclerosis, were negative for anti-Scl-70 antibody by the more specific immunodiffusion method of testing. METHODS: Patients evaluated by a rheumatologist at a Scleroderma referral center who had tested positive for anti-Scl-70 antibody prior to referral, but did not have classifiable SSc based on clinical criteria, were invited to undergo testing for anti-Scl-70 antibody by immunodiffusion. Patient demographics and clinical features were recorded at the time of their evaluation, and diagnostic testing results were reviewed using the medical records. RESULTS: 52 patients were enrolled over an 8-year period, with 48 (92.3%) testing negative and 4 (7.7%) testing positive for anti-Scl-70 antibody by immunodiffusion. Of the 48 patients who tested negative, 18 (37.5%) tested negative for ANA by indirect immunofluorescence, 33 (68.8%) did not have Raynaud's phenomenon, and 43 (89.6%) had ≤1 clinical criteria items based on the 2013 ACR/EULAR SSc classification criteria. Nevertheless, 21 (43.8%) patients who were negative for anti-Scl-70 antibody by immunodiffusion had undergone a chest CT and 14 (29.2%) had undergone an echocardiogram. A total of 23 patients had at least one follow up clinic visit. 3 out of 4 patients who were positive for anti-Scl-70 antibody by immunodiffusion, but none of the 19 patients who tested negative by immunodiffusion, developed sufficient criteria during follow up to be classified as SSc. CONCLUSION: Assays for anti-Scl-70 antibody in commercial laboratories that are commonly utilized in clinical practice can produce false positive results. These results can lead to angst for patients, as well as unnecessary referrals and diagnostic evaluations.

First record of antibodies to the bluetongue virus in ewe (Ovis aries) in the state of Amazonas, Brazil / Primeiro registro de anticorpos para o vírus da língua azul em ovino (Ovis aries) no estado do Amazonas, Brasil

The objective of this work was to describe the first record of antibodies to the Bluetongue Virus (BTV) in ewe, in the state of Amazonas. The ewe, which was in twin pregnancy, gave birth on May 9, 2015, but a lamb died hours after delivery. Veterinary service was then requested by the owner, where emaciation, loss of wool, pyrexia, apathy, dyspnea, mucoid nasal secretion, facial, lingual and submandibular edema were observed. There was a visit by the Agricultural Defense Agency of the State of Amazonas to the property and blood samples were collected from the animal. The whole blood and serum were sent to the National Agricultural Laboratory, where it was possible to detect the presence of specific antibodies to BTV, through the Agar Gel Double Immunodiffusion. The ewe was submitted to a new blood collection, following the same protocols and the samples were sent to the Biological Institute of São Paulo, confirmed diagnosis. The animal in a serious clinical condition, could not resist and died in July 2015. The occurrence of an allochthonous case, in an area where vector insects occur, can trigger an endemic process in the Amazon region. With this, the epidemiological control of these occurrences is necessary, in order to avoid the spread of the disease in the country.

O objetivo do trabalho foi descrever o primeiro registro de anticorpos para o Vírus da Língua Azul (VLA) em ovino, no estado do Amazonas. A ovelha, que se encontrava em gestação gemelar, pariu no dia 9 de maio de 2015, porém um cordeiro faleceu horas após o parto. Foi então solicitado serviço veterinário por parte do proprietário, onde foi observado emaciação, perda de lã, pirexia, apatia, dispneia, secreção nasal mucoide, edema facial, lingual e submandibular. Houve visita da Agência de Defesa Agropecuária do Estado do Amazonas na propriedade e coletadas amostras de sangue do animal. O sangue total e soro foram enviados ao Laboratório Nacional Agropecuário, no qual foi possível detectar a presença de anticorpos específicos para VLA, através do teste de Imunodifusão Dupla em Gel de Ágar. A ovelha foi submetida a uma nova coleta de sangue, seguindo os mesmos protocolos e as amostras foram enviadas ao Instituto Biológico de São Paulo, confirmando diagnóstico. O animal em estado clínico grave, não resistiu e veio a óbito em julho de 2015. A ocorrência de um caso alóctone, em uma área de ocorrência de insetos vetores, pode desencadear um processo de endemia na região amazônica. Com isso, o controle epidemiológico destas ocorrências, se fazem necessários, afim de se evitar a disseminação da doença no país.