Antigenic Fractions Research Articles

Indirect ELISAs with sucrose subcellular fractions of Neospora caninum as antigens for diagnosis of neosporosis in cattle

Neosporosis is one of the major causes of abortion in cattle, and it is responsible for significant economic losses in those animals. Thus, this study aimed to evaluate indirect ELISA using subcellular fractions of Neospora caninum obtained via sucrose gradient separation. Eighty-five sera from dairy cattle previously tested using indirect immunofluorescence assay (IFA) were used. Three distinct bands were separated at 1.0 M, 1.4 M, 1.6 M, and the pellet at 1.8 M, which were identified as fractions one (F1), two (F2), three (F3), and four (F4), respectively. These fractions showed parasite membranes in the F1, rhoptry and conoids in the F2, mitochondria in the F3, and tachyzoite ghosts remain in F4. Indirect ELISAs for IgM, and IgG were performed. Additionally, sensitivity, specificity, and kappa values were defined considering the IFA as the gold standard. The highest and lowest specificities were observed for F1 (76 %) and F3 (16 %), respectively. F2 and F4 showed the highest sensitivity (93.3 %), kappa agreement (0.46), and Negative Preventive Value (NPV) (73 %) respectively. It was possible to standardize indirect ELISAs using whole soluble antigen and subcellular fractions of N. caninum, and F2 and F4 showed higher sensitivity (93.3 %), kappa (0.41), and NPV values (75 %) than F1, and F3, which could be used for epidemiology studies such as screening.

Increased specificity of Fasciola hepatica excretory-secretory antigens combining negative selection on hydroxyapatite and salt precipitation

A single and rapid method to obtain an antigenic fraction of excretory-secretory antigens (ESAs) from Fasciola hepatica suitable for serodiagnosis of fascioliasis is reported. The procedure consists in the negative selection of F. hepatica ESAs by hydroxyapatite (HA) chromatography (HAC; fraction HAC-NR) followed by antigen precipitation with 50% ammonium sulphate (AS) and subsequent recovery by means of a Millex-GV or equivalent filter (Fi-SOLE fraction). Tested in indirect ELISA, the Fi-SOLE antigens detected natural infections by F. hepatica with 100% sensitivity and 98.9% specificity in sheep, and 97.7% sensitivity and 97.7% specificity in cattle, as determined by ROC analysis. The SDS-PAGE and proteomic nano-UHPLC-Tims-QTOF MS/MS analysis of fractions showed that the relative abundance of L-cathepsins and fragments thereof was 57% in fraction HAC-NR and 93.8% in fraction Fi-SOLE. The second most abundant proteins in fraction HAC-NR were fatty-acid binding proteins (11.9%). In contrast, free heme, and heme:MF6p/FhHDM-1 complexes remained strongly bond to the HA particles during HAC. Interestingly, phosphorylcholine (PC)-bearing antigens, which are a frequent source of cross-reactivity, were detected with an anti-PC mAb (BH8) in ESAs and fraction HAC-NR but were almost absent in fraction Fi-SOLE.

BIOM-56. THE PRIVATE AND RECURRING ANTIGENIC PROFILE OF MOST AGGRESIVE PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Efficient immunotherapies for pediatric brain tumors face a significant challenge in identifying tumor rejection antigens. To overcome this hurdle, we utilized a comprehensive computer algorithm called the Open Reading Frame Antigen Network (O.R.A.N.). METHODS Our focus was on expressed epitopes, including single nucleotide variations, small indels, gene fusions, novel junctions from alternative splicing, and Tumor Associated Antigens (TAAs), for antigenic epitope predictions. To ensure uniqueness, we compared immunogenic TAA epitopes against a personalized human proteomic library, ensuring they were not shared by other expressed isoforms or genes. The immunological landscape of medulloblastoma (n = 170) and DIPG (n = 30) was explored using immune deconvolution techniques combined with single-cell RNA sequencing integration. RESULTS Our hypothesis was that medulloblastomas and DIPG exhibit diverse immune profiles and express genes that are rarely found in normal tissues, making them potential targets for immunotherapy. In this study, we found that most neoantigens identified in medulloblastoma patients were unique to each individual, emphasizing the need for personalized neoantigen-based therapies. Interestingly, recurring neoantigens were predominantly present in the MB SHH subgroup, while they constituted only a small fraction of putative antigens in WNT, Group 3, and Group 4 patients. On the other hand, recurring Tumor Associated Antigens (TAAs) were more frequently expressed in the latter three molecular subgroups but not in the SHH subgroup. Thus, a combination of personalized neoantigen vaccines and targeting recurring TAAs could potentially maximize the anti-tumor effects. CONCLUSION Medulloblastoma is a disease characterized by its heterogeneity, and current standard treatments do not adequately address this aspect. In the case of DIPG, surgical resection is not a viable option due to its diffuse and infiltrative nature within the brainstem. By leveraging the power of the immune system, cellular immunotherapy holds promise in providing more targeted and personalized treatment options for pediatric brain tumor patients.

Molecular Characterization of the Interplay between Fasciola hepatica Juveniles and Laminin as a Mechanism to Adhere to and Break through the Host Intestinal Wall.

Fasciola hepatica is the main causative agent of fasciolosis, a zoonotic parasitic disease of growing public health concern. F. hepatica metacercariae are ingested by the host and excyst in the intestine, thereby releasing the newly excysted juveniles (FhNEJ), which traverse the gut wall and migrate towards the biliary ducts. Since blocking F. hepatica development is challenging after crossing of the intestinal wall, targeting this first step of migration might result in increased therapeutic success. The intestinal extracellular matrix (ECM) is constituted by a network of structural proteins, including laminin (LM) and fibronectin (FN), that provide mechanical support while acting as physical barrier against intestinal pathogens. Here, we employed ELISA and immunofluorescent assays to test for the presence of LM- and FN-binding proteins on a tegument-enriched antigenic fraction of FhNEJ, and further determined their identity by two-dimensional electrophoresis coupled to mass spectrometry. Additionally, we performed enzymatic assays that revealed for the first time the capability of the juvenile-specific cathepsin L3 to degrade LM, and that LM degradation by FhNEJ proteins is further potentiated in the presence of host plasminogen. Finally, a proteomic analysis showed that the interaction with LM triggers protein changes in FhNEJ that may be relevant for parasite growth and adaptation inside the mammalian host. Altogether, our study provides valuable insights into the molecular interplay between FhNEJ and the intestinal ECM, which may lead to the identification of targetable candidates for the development of more effective control strategies against fasciolosis.

Direct Quantification of Protein Antigens in Subunit Plague and Rickettsial Vaccine Preparations

The aim of the work was to put forward the methods for direct quantitative determination of the content of Yersinia pestis and Rickettsia raoultii protein antigens in preparations and various prototypes of subunit vaccines. Materials and methods. Y. pestis LcrV and Caf1 antigens enclosed in the substance of the molecular microencapsulated plague vaccine (MMPV) and separately, in microcrystals of amino acids co-precipitated with plague proteins were used as model antigens. R. raoultii Adr2, OmpB24, and YbgF antigens were adsorbed on the prototype substance of the rickettsia vaccine. The release of plague antigens from MMPV microcapsules was carried out through successive treatment of the latter with organic solvents, methylene chloride and methanol, respectively; the carrier microcrystals were dissolved in 0.1 M sodium citrate buffer at pH 6.0. The antigen content in the prototype substance of the rickettsial vaccine was determined by measuring the amount of proteins not bound to the alumogel. Quantitative parameters characterizing the content of antigens in the substances and prototypes of vaccine preparations were calculated by processing digital images of polyacrylamide gels obtained by electrophoresis of protein antigen fractions extracted from carriers. Results and discussion. Methods for direct extraction and subsequent quantitative analysis of Y. pestis LcrV and Caf1 antigens from subunit vaccine preparations based on amino acid microcrystals and polylactide microcapsules that do not cause protein degradation have been studied. A different nature of the binding of LcrV and Caf1 in the substances of microcrystals has been established, while the proportion of antigens released from microcrystals has been quantified only in case of their complete dissolution. It was found that at low concentrations of LcrV and Caf1 proteins extracted from microcrystals, it is necessary to concentrate the extracts with subsequent removal of salts for their reliable visualization. It has been confirmed that 10 μg of plague antigens and proteins of R. raoultii in a dose volume of 200 μl of suspension is sufficient for quantitative analysis using electrophoretic method. The prospects of other physicochemical methods alternative to direct extraction of antigens for evaluating the composition and quality of vaccine preparations are discussed.

Development of an In House Enzyme-Linked Immunosorbent Assay (ELISA) Prototype Using Different Anti- gens for Serological Diagnosis of Brucella ovis

Bu çalışmada, Brucella ovis’e karşı humoral yanıtın değerlendirilmesinde kullanılmak üzere, bu bakterinin farklı antijenik fraksiyonlarından hazırlanan antijenlerin karşılaştırılmasıyla bir Enzyme -Linked Immunosorbent Assay (ELISA) prototipinin geliştirilmesi amaçlandı. Bu amaçla, dört farklı antijen (Hot salin ekstrakt, Rough Lipopolisakkarit, Faj lizat 1 ve Faj lizat 2), 84 koyun serum örneği ile test edildi. Brucella ovis açısından ELISA seropozitiflikleri, kullanı- lan antijenlere göre sırasıyla %14.3, %13.1, %10.7 ve %15.5 olarak saptandı. En yüksek pozitiflik oranı Faj lizat 2 ile elde edildi, ancak, Brucella ovis’e karşı oluşan antikorların ELISA ile belirlenmesi için kullanılan 4 farklı antijenin test sonuçları arasındaki farklılık, istatistiki açıdan önemsiz (χ2=0.89; P>0.05) bulundu. Sonuç olarak, gelecekteki çalışma- larda, Faj lizat 2 antijeni kullanılarak ELISA ile test edilen çok daha fazla serum örneği için elde edilen sonuçların, komplement fikzasyon testi ve agar jel immunodifüzyon testi gibi standart testler ve temin edilebilecek ticari kitlerin sonuçları ile karşılaştırılmasına ihtiyaç duyulacağı ve böylece in house ELISA prototipinin ticarileşme potansiyelinin daha sağlıklı olarak değerlendirilebileceği kanısına varıldı. Ayrıca, az sayıda serum örneğiyle elde edilen seropozitiflik oranı göz önünde bulundurulduğunda, Türkiye'nin farklı bölgelerini kapsayan daha fazla sayıda serum örneği ile geniş ölçekli bir serolojik çalışmanın yapılması ile ülkemizdeki gerçek hastalık durumunun ortaya çıkarılabileceği düşünül- mektedir.

Effects of adult Ascaris suum and their antigens (total and trans-cuticular excretory-secretory antigen, cuticular somatic antigen) on intestinal nutrient transport in vivo.

Ascaris suum constitutes a major problem in commercial pig farming worldwide. Lower weight gains in infected pigs probably result from impaired nutrient absorption. This study investigated intestinal nutrient transport in 4 groups of 6 pigs each, which were inoculated with 30 living adult A. suum, or antigen fractions consisting of (1) total excretory–secretory (ES) antigens of adult worms, (2) ES antigens secreted exclusively from the parasites' body surface (trans-cuticular ES) and (3) cuticular somatic antigens of A. suum, compared to placebo-treated controls. Three days after inoculation into the gastrointestinal tract, glucose, alanine and glycyl-l-glutamine transport was measured in the duodenum, jejunum and ileum using Ussing chambers. Transcription of relevant genes [sodium glucose cotransporter 1 (SGLT1), glucose transporter 1 (GLUT1), GLUT2, hypoxia-inducible factor 1-alpha (Hif1α), interleukin-4 (IL-4), IL-13, signal transducer and activator of transcription 6 (STAT6), peptide transporter 1 (PepT1)] and expression of transport proteins [SGLT1, phosphorylated SGLT1, GLUT2, Na+/K+-ATPase, amino acid transporter A (ASCT1), PepT1] were studied. Although no significant functional changes were noted after exposure to adult A. suum, a significant downregulation of jejunal GLUT1, STAT6, Hif1α and PepT1 transcription as well as ileal GLUT2 and PepT1 expression indicates a negative impact of infection on transport physiology. Therefore, the exposure period of 3 days may have been insufficient for functional alterations to become apparent. In contrast, A. suum antigens mainly induced an upregulation of transport processes and an increase in transcription of relevant genes in the duodenum and jejunum, possibly as a compensatory reaction after a transient downregulation. In the ileum, a consistent pattern of downregulation was observed in all inoculated groups, in line with the hypothesis of impaired nutrient transport.

Changing Landscape of Cancer Vaccines-Novel Proteomics Platform for New Antigen Compositions.

The creation of cancer vaccines is a constant priority for research and biotechnology. Therefore, the emergence of any new technology in this field is a significant event, especially because previous technologies have not yielded results. Recently, the development of a cancer vaccine has been complemented by a new proteomics technology platform that allows the creation of antigen compositions known as antigenic essences. Antigenic essence comprises a target fraction of cellular antigens, the composition of which is precisely controlled by peptide mass spectrometry and compared to the proteomic footprint of the target cells to ensure similarity. This proteomics platform offers potential for a massive upgrade of conventional cellular cancer vaccines. Antigenic essences have the same mechanism of action, but without the disadvantages, and with notable advantages such as precise targeting of the immune response, safety, controlled composition, improved immunogenicity, addressed MHC restriction, and extended range of vaccination doses. The present paper calls attention to this novel platform, stimulates discussion of the role of antigenic essence in vaccine development, and consolidates academic science with biotech capabilities. A brief description of the platform, list of cellular cancer vaccines suitable for the upgrade, main recommendations, limitations, and legal and ethical aspects of vaccine upgrade are reported here.

Chemokine production induced by Corynebacterium pseudotuberculosis in a murine model.

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis. The main clinical sign of this disease is the development of granulomas, especially in small ruminants; however, the pathways that are involved in the formation and maintenance of these granulomas are unknown. Cytokines and chemokines are responsible for the migration of immune cells to specific sites and tissues; therefore, it is possible that chemokines participate in abscess formation. This study aimed to evaluate the induction of chemokine production by two C. pseudotuberculosis strains in a murine model. A highly pathogenic (VD57) and an attenuated (T1) strain of C. pseudotuberculosis, as well as somatic and secreted antigens derived from these strains, was used to stimulate murine splenocytes. Then, the concentrations of the chemokines CCL-2, CCL-3, CCL-4, and CCL-5 and the cytokines IL-1 and TNF were measured in the culture supernatants. The VD57 strain had a higher ability to stimulate the production of chemokines when compared to T1 strain, especially in the early stages of stimulation, which can have an impact on granuloma formation. The T1 lysate antigen was able to stimulate most of the chemokines studied herein when compared to the other antigenic fractions of both strains. These results indicate that C. pseudotuberculosis is a chemokine production inducer, and the bacterial strains differ in their induction pattern, a situation that can be related to the specific behavior of each strain.

Toxocara canis 30-35 kDa excretory-secretory antigen is an important marker in mice challenged by inocula containing different parasite load levels.

ABSTRACTThe Western-blotting technique was applied to identify antigenic fractions of excretory-secretory Toxocara canis antigen recognized by IgG antibodies throughout an experimental infection in mice challenged by different inocula. Mice were inoculated with 5, 50 and 500 embryonated eggs and serum samples were collected 15, 30, 60, 90 and 120 days post-infection. Serum samples were analyzed using an excretory-secretory Toxocara antigen. Antibodies recognized antigenic fractions from 30 to 90 kDa. The protein fraction of 30-35 kDa was the most frequently recognized regardless of the size of inoculum and the stage of infection represented by the different collection times, but the antigenic recognition was more evident in groups infected with 50 and 500 eggs. This study presents an antigenic panel of the excretory-secretory antigen of T. canis and suggests that the 30-35 kDa antigenic fraction is a promising marker of the infection and should be further explored in future studies on experimental toxocariasis.

ПРЕПАРАТИВНОЕ ВЫДЕЛЕНИЕ СПЕЦИФИЧЕСКИХ АНТИГЕНОВ ПОЛНОГО КЛЕТОЧНОГО ЛИЗАТА M. BOVIS.

Bovine tuberculosis is a serious problem for agriculture all over the world and causes huge economic losses. At the same time, infections caused by Mycobacterium bovis not only affect animal husbandry and the food industry, but also pose a threat to public health, as M. bovis is often isolated as a pathogen of tuberculosis, especially in patients with various immune deficiencies. The situation is aggravated by the lack of simple and accessible methods for diagnosing tuberculosis in cattle, which significantly impedes the monitoring of infection and the application of appropriate preventive measures. Serological tests, due to their relatively cheap cost, rapidity, rather high sensitivity and specificity, are able to compete with the allergic test during mass examinations of cows. In the present study, we present the results on the study of the antigenic structure of mycobacteria and the isolation of the most specific antigenic components from the lysate of the M. Bovis cell wall destroyed on the Fast Prep 24 device. The developed method allows obtaining a serologically active antigenic fraction with a high degree of purity with a molecular weight of 28 kDa.

Isolation and Purification of Low Molecular Weight Proteins from Culture Filtrate of Mycobacterium Tuberculosis Strain C.

In the last couple of years, a number of new and rapid tests for the diagnosis of Tuberculosis (TB) have been developed based on the low molecular weight antigens from Mycobacterium tuberculosis (Mtb) culture supernatant. This study aimed to isolate and purify low molecular weight antigens secreted by Mtb strain C for diagnostic purpose. The secretory proteins from culture filtrate of Mtb were extracted using ammonium sulphate precipitations and sephadex-G50 gel chromatography. The obtained antigen fractions were analyzed for their protein concentrations and approximate molecular weight using Lowry method and SDS-PAGE (12.5%), respectively. DOT-ELISA and Western blot assay was performed to confirm the presence of purified low molecular weight proteins isolated from Mtb using sera from pulmonary tuberculosis patients (polyclonal antibodies). During chromatography, low molecular weight proteins were separated, that was approximately 0.7 mg/ml of the total proteins (1.662 mg/ml). The purified protein fractions in molecular weight range of 14 kDa-41kDa appeared during SDS-PAGE analysis. The chromatographic band fraction in the weight range of 30-41 kDa was identified in the TB patients’ sera using Western blotting. The low molecular weight proteins in the culture filtrate of Mtb strain C were purified using ammonium sulphate and chromatography. These fractions were confirmed using Western blotting. The obtained results might support the hypothesis that the Mtb culture filtrate antigens could be used as a rapid and sensitive assay for the detection of patients with pulmonary TB.

Immunogenicity in Oreochromis niloticus vaccinated with sonicated antigens against streptococcosis

Streptococcosis causes great economic losses in intensive culture of tilapia. Vaccination is the most effective and safest way to tackle infectious diseases. Thus, this study sought the more effective and safer antigenic fraction after sonication of Streptococcus agalactiae to elaborate a vaccine against streptococcosis in Nile tilapia. For this, twenty-one days after vaccination with different fractions (soluble and insoluble) of S. agalactiae, the fish were challenged with the homologous strain (LD50). Then, samples were taken at zero, 14, 28, 60 and 90 days post-vaccination (DPV, n = 7). Blood and organs (cranial kidney, spleen and liver) were collected from vaccinated and unvaccinated fish. Finally, insoluble fraction vaccine presented the best effect, resulting in a 100% relative percent of survival (RPS) and without clinical manifestations. In view of the results, it was to evaluate the role of the insoluble fraction of the antigen in the protective immunity against streptococcosis. The results indicate that the spleen might be the main organ in the vaccine response in Nile tilapia due to the great morphological and immunological differences in vaccinated fish, evidenced by the greater of melanomacrophage centers (MMC) and IgM + lymphocytes in relation to the non-vaccinated fish. At 60 DPV, it was observed the peak of the protective immunity related to the maximum concentration of proteins, circulating leukocytes, antibody titers in the serum and tissue changes with greater expression of IgM + and MMC number in the spleen and kidney of Oreochromis niloticus. Vaccination with insoluble fraction of S. agalactiae was safe and provided effective protection against streptococcosis with maximum protective response at 60 DPV.

Efficacy of a combination modified-live IBR-BVD-PI3-BRSV vaccine + Mannheimia haemolytica toxoid against challenge with virulent BVDV-1b and BVDV-2 viruses in young calves 60 days of age

Efficacy of attenuated (att) bovine viral diarrhea virus (types 1 and 2) as antigen fractions in a modified-live multivalent vaccine were evaluated following single, subcutaneous (SC) administration and intranasal (IN) challenge 35 to 38 d after vaccination, with either virulent BVDV-1b (Study 1) or BVDV-2 (Study 2) viruses in young calves. A total of 80 BVDV-seronegative Holstein calves, 53 to 61 d of age at the time of vaccination, were used in 2 separate studies with 40 animals per study. In each study, calves were allocated to 1 of 2 treatment groups (20 animals per group) which received either a single dose of combination modified-live bovine rhinotracheitis (BHV-1)-bovine virus diarrhea-parainfluenza 3-respiratory syncytial virus vaccine + Mannheimia haemolytica toxoid, or corresponding placebo formulation without targeted test antigen fractions attBVDV-1a and attBVDV-2. In the respective studies, multivalent vaccine induced significantly higher virus neutralizing antibody responses and reduced incidence and duration of leukopenia and viremia in vaccinated animals compared to placebo-treated animals. Post-challenge leukopenia, a hallmark of BVDV infection, was observed in 75% and 100% of control calves compared to only 26.3% and 25% in vaccinated animals in Study 1 and Study 2, respectively (p=0.006; p=0.0001). In addition, duration of leukopenia was significantly shorter in vaccinates compared to placebo controls (p=0.0091 Study 1; p<0.0001 Study 2). Furthermore, 100% of placebo-treated calves in both studies were viremic compared to 57.9% (Study 1) and 25% (Study 2) of vaccinated animals, resulting in significant reduction of post-challenge viremia (p=0.0012, Study 1; p=0.0001, Study 2). The duration of viremia was significantly shorter (p<0.0001) in vaccinated groups compared to control calves in both studies. In conclusion, data from the current studies demonstrated vaccine efficacy in 60-day-old calves against BVDV-1b and BVDV-2 infection.

Immunological detection of human and camel cystic echinococcosis using different antigens of hydatid cyst fluid, protoscoleces, and germinal layers.

Background and Aim:Cystic echinococcosis (CE)/hydatidosis is one of the most prevalent neglected zoonotic diseases. It is initially asymptomatic and does not produce any clinical signs until the cyst becomes enlarged, causing localized pressure on internal organs and tissues. Therefore, the detection of Echinococcus granulosus antibodies is highly essential. This study evaluated the antigens of hydatid cyst fluid, protoscoleces, and germinal layers for efficient immunological diagnosis of CE in humans and camels.Materials and Methods:Hydatid cyst fluid (FLc), protoscoleces (Psc), and the germinal layer (GLc) antigens were prepared from camel-lung hydatid cysts. In the same way, hydatid cyst fluid (FLh) and protoscoleces (Psh) antigens from human-liver cyst aspirate were produced. The comparative immunodiagnostic efficacy of the prepared antigens was verified using indirect enzyme-linked immunosorbent assay (ELISA), SDS-PAGE, and immunoblotting.Results:ELISA proves that FLc and GLc antigens were higher than FLh and Psh antigens. This shows that binding reactivity in naturally infected human sera, camel sera, and Psc is the most potent, exhibiting 100% sensitivity with 78.26% and 76.47% specificity in camel and human sera, respectively. The CE prevalence using diagnostic Psc was 54.79% and 61.32% in tested human and camel sera, respectively. The electrophoretic profiles of all shared antigens showed similarities at 52, 41, and 22 kDa. Immunoblotting demonstrated common immune-reactive bands in all antigen types at 52 and 41 kDa against positive human and camel sera.Conclusion:This immunological study introduces camel hydatid cyst Psc as a potent diagnostic antigen and new immune-reactive fractions of 52 and 41 kDa for diagnosing hydatidosis in humans and camels.

Efficacy of a novel intradermal Lawsonia intracellularis vaccine in pigs against experimental infection and under field conditions

BackgroundThe efficacy of a novel inactivated intradermal Lawsonia intracellularis vaccine, Porcilis® Lawsonia ID, was evaluated in two experimental vaccination-challenge studies and under field conditions on a farm with a history of recurrent acute ileitis. In addition, the efficacy of the vaccine was compared to that of a commercially available live attenuated vaccine. The novel inactivated vaccine consists of a freeze-dried antigen fraction that is dissolved just prior to use in either the adjuvant or in Porcilis® PCV ID; an existing intradermal vaccine against porcine Circovirus type 2. In the two experimental vaccination-challenge studies, groups of 25 piglets were vaccinated once at 3 weeks of age or left unvaccinated as challenge control. Vaccines tested were Porcilis® Lawsonia ID as standalone (study 1) or in associated mixed use with Porcilis® PCV ID (study 2) and an orally administered commercially available live vaccine (study 1). The pigs were challenged with virulent L. intracellularis at 4 weeks (study 1) or 21 weeks (study 2) after vaccination. Post-challenge, the pigs were evaluated for clinical signs, average daily weight gain, shedding and macroscopic as well as microscopic immuno-histological ileum lesion scores. In the field study, the mortality and key performance parameters were evaluated over a period of 8 months.ResultsThe results of the two experimental vaccination-challenge studies showed that Porcilis® Lawsonia ID as single vaccine or in associated mixed use with Porcilis® PCV ID, induced statistically significant protection against experimental L. intracellularis infection, 4 weeks or 21 weeks after vaccination. This was demonstrated by lower clinical scores, improved weight gain, reduction of L. intracellularis shedding and reduction of macroscopic as well as microscopic ileum lesion scores when compared to the controls. The protection induced was superior to that of the commercially available live vaccine. In the field study Porcilis® Lawsonia ID was highly efficacious in reducing L. intracellularis associated mortality and improving key production parameters.ConclusionThe results support that this new intradermal vaccine is efficacious against L. intracellularis and may be used in associated mixed use with Porcilis® PCV ID.

Efficacy of a modified-live IBR-BVD-PI3-BRSV-Mannheimia haemolytica toxoid vaccine against challenge with virulent BHV-1 and PI3 viruses in calves 60 days of age

Efficacy of attenuated (att) bovine herpesvirus type 1 (BHV-1) and bovine parainfluenza type-3 (PI3) viruses as antigen fractions in a modified-live virus (MLV) multivalent vaccine was evaluated following single, subcutaneous (SC) administration and intranasal (IN) challenge 28 days after vaccination, with either virulent BHV-1 or PI3 viruses in young calves. A total of 80 seronegative calves, 50 to 63 days of age at the time of vaccination, were used in 2 separate studies with 40 animals per study. Calves were allocated to 2 treatment groups with 20 animals per group which received either a single dose of a MLV IBR-BVD-PI3-BRSV vaccine, Mannheimia haemolytica toxoid, or corresponding placebo formulation without targeted test antigen fractions attBHV-1 (Study 1) or attPI3 (Study 2). Incidence and duration of clinical signs associated with respiratory disease, rectal temperatures, virus shedding, and serologic responses were compared between treatment groups in each study to assess vaccine efficacy. The 5-way vaccine induced significantly higher (p<0.0001) virus neutralizing antibody responses and reduction in fever (p<0.0001), mean rectal temperatures, and lower incidence or shorter duration of clinical disease related to BHV-1 and PI3 infection than placebo-treated calves. Infectious bovine rhinotracheitis (IBR) disease, hallmark of BHV-1 infection, was observed in 95% of control calves and in only 10% of vaccinates. Vaccinated animals in Study 1 and Study 2 demonstrated a 98.8 and 98.9% reduction in virus shedding, respectively, and significantly (p<0.0001) shorter duration of virus shedding compared to control calves, demonstrating protective vaccine efficacy.

A simple and efficient purification method of native immunoreactive antigen for diagnosis of camel hydatidosis.

Background:Cystic echinococcosis (CE), a zoonotic disease that affects animal and human health, is of increasing economic importance due to high morbidity rates and high economic losses in the livestock industry.Aim:The present study was conducted to purify the antigen from hydatid cyst fluid (HCF) with high diagnostic efficacy of camel hydatidosis using indirect enzyme-linked immunosorbent assay (ELISA).Materials and Methods:The HCF antigen was purified using Sephacryl S-300 column chromatography. Characterization of fractions was performed using reducing and non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Further, antibodies against Echinococcus granulosus cysts in camel serum were detected using indirect ELISA.Results:The purification process resulted in three fractions of antigens: FI, FII, and FIII. Indirect ELISA showed that higher diagnostic efficacy was observed in FI than in FII and FIII. Indirect ELISA, in which FI was utilized, showed 88% sensitivity and 91.7% specificity. Non-reducing SDS-PAGE showed that FI had two bands of molecular weights 120 and 60 kDa. Western blot analysis of FI demonstrated that 60, 38, and 22 kDa were antigenic bands when reacted with naturally infected camel sera with E. granulosus cysts. Using indirect ELISA, F1 recorded an infection percentage of 81.7% in randomly collected camel serum samples.Conclusion:FI is a promising antigen for accurate diagnosis of camel CE using indirect ELISA.

Immunoreactivity of proteins within 30-40 kDa range during the acute and the recovery phases in rats experimentally infected with Strongyloides venezuelensis.

ABSTRACTIn experimental infection with Strongyloides venezuelensis, the acute and recovery phases can be distinguished, unlike human infections caused by Strongyloides stercoralis. The objective of this study was to evaluate the production of anti-Strongyloides IgG antibodies and the recognition of immunogenic protein bands during the acute and the recovery phases in rats experimentally infected with S. venezuelensis. Rats were infected subcutaneously with 400 or 4,000 S. venezuelensis infective larvae. The acute phase was characterized by elimination of a large number of eggs in the faeces on days 6-14 post infection; the recovery phase was characterized by the resolution of the infection between days 30 and 35 post infection. Differences in IgG levels were observed in the acute and the recovery phases. Different antigenic fractions were recognized in both phases of infection. It is concluded that proteins within the 30-40 kDa range are immunoreactive markers for both the acute and the recovery phases in rats experimentally infected with S. venezuelensis, particularly using membrane antigen.

Review of Free Prostate-Specific Antigen Assay Utilization at Danbury Hospital

Abstract Introduction Measuring the concentration of free fraction of prostate-specific antigen (fPSA) present in serum in addition to the total PSA (tPSA) concentration has been shown to enhance specificity over tPSA alone in evaluating male patients with mild-moderate elevations of tPSA in the diagnostic “gray zone” (4.0-10.0 ng/mL) and negative findings on digital rectal exam. The clinical utility of fPSA has not been demonstrated outside the tPSA gray zone, as the probability of prostate cancer is very low at tPSA <4.0 and very high at tPSA >10.0. However, many clinicians order tPSA and fPSA concurrently without first knowing the results of the tPSA. This study was to review orders for fPSA and provide suggestions for improving utilization. Methods Paired test results for concurrently ordered tPSA and fPSA tests during the time periods of January to February 2018 and January to February 2019 (before and after laboratory information system upgrade) were extracted for quality assurance purposes. No patient identifiers were included. Data were sorted and the percentage of “appropriate” fPSA orders was calculated for each time period. Results The number of concurrent total and fPSA tests ordered was 174 for January-February 2018 and 232 for January-February 2019. The number of tPSA within the 4.0 to 10.0 ng/mL range was 78 for January-February 2018 and 148 for January-February 2019. The percentage of “appropriate” fPSA test orders (tPSA 4.0-10.0 ng/mL) was 44.8% for January-February 2018 and 63.8% for January-February 2019. Conclusion A high percentage of inappropriate fPSA orders was found during both time periods. Inappropriate fPSA testing is not only unnecessary and wasteful but can lead to diagnostic errors. Options for driving improved utilization include automatic canceling of fPSA orders when tPSA is not within the diagnostic gray zone and offering fPSA as a “reflex” test that is performed only when tPSA is within the diagnostic gray zone.